WO2021125805A1 - Kit for preparing nanoparticle composition for drug delivery, comprising polylactic acid salt - Google Patents

Kit for preparing nanoparticle composition for drug delivery, comprising polylactic acid salt Download PDF

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WO2021125805A1
WO2021125805A1 PCT/KR2020/018480 KR2020018480W WO2021125805A1 WO 2021125805 A1 WO2021125805 A1 WO 2021125805A1 KR 2020018480 W KR2020018480 W KR 2020018480W WO 2021125805 A1 WO2021125805 A1 WO 2021125805A1
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weight
kit
nanoparticles
parts
chamber
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PCT/KR2020/018480
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French (fr)
Korean (ko)
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이소진
김상훈
박준영
남혜영
손지연
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주식회사 삼양홀딩스
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Priority to US17/787,156 priority Critical patent/US20230039124A1/en
Priority to BR112022012118A priority patent/BR112022012118A2/en
Priority to JP2022538147A priority patent/JP2023508313A/en
Priority to AU2020407348A priority patent/AU2020407348A1/en
Priority to CA3162374A priority patent/CA3162374A1/en
Priority to EP20901243.4A priority patent/EP4079298A4/en
Priority to CN202080096831.7A priority patent/CN115135310A/en
Publication of WO2021125805A1 publication Critical patent/WO2021125805A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/186—Quaternary ammonium compounds, e.g. benzalkonium chloride or cetrimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present invention relates to a kit for preparing a nanoparticle composition for drug delivery, and more specifically, a drug can be prepared by simply mixing the amphiphilic block copolymer, cationic compound, polylactate, and drug, which are kit components. It relates to a kit for preparing a nanoparticle composition for drug delivery designed to easily form nanoparticles encapsulated therein.
  • the delivery system is largely divided into a viral delivery system using adenovirus or retrovirus, etc. and a non-viral delivery system using cationic lipids and cationic polymers.
  • a viral carrier it is known that there are many problems in commercialization because it is exposed to risks such as non-specific immune reaction and the production process is complicated. Therefore, the recent research direction is in the direction of improving the shortcomings by using a non-viral carrier.
  • the non-viral delivery system has the advantages of fewer side effects in terms of in vivo safety and a low production price in terms of economic feasibility.
  • Non-viral carriers used for the delivery of nucleic acid substances are a complex (lipoplex) of a cationic lipid and a nucleic acid using a cationic lipid, and a complex (polyplex) of a polycationic polymer and a nucleic acid.
  • cationic lipids or polycationic polymers have been studied in that they stabilize the anionic drug by forming a complex through electrostatic interaction with the anionic drug and increase intracellular delivery (De Paula D) , Bentley MV, Mahato RI, Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting, RNA 13 (2007) 431-56; Gary DJ, Puri N, Won YY, Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer -based DNA delivery, J Control release 121 (2007) 64-73).
  • the nanoparticles formed by these complexes often lose stability easily depending on the storage environment, so they are vulnerable to long-term storage and there is a risk that the quality may be damaged during transportation.
  • it is very difficult to manufacture because it requires a complicated manufacturing process to ensure sufficient stability.
  • An object of the present invention is to easily form drug-containing nanoparticles by simply mixing the kit components so that it is easy for the end consumer to use, and drug-containing nanoparticles can be easily formed immediately before use.
  • An object of the present invention is to provide a kit for preparing a nanoparticle composition for drug delivery, which can effectively deliver a drug into the body without the influence of storage or transportation environment.
  • the present invention provides a first chamber containing an amphiphilic block copolymer, a cationic compound, and a polylactate; and a second chamber containing an active ingredient selected from a nucleic acid, a polypeptide, a virus, or a combination thereof; provides a kit for preparing a nanoparticle composition comprising a.
  • the kit is for forming nanoparticles that deliver an intracellular active ingredient.
  • At least one selected from the group consisting of the first chamber and the second chamber further includes an additional solvent.
  • the solvent is an aqueous solvent, a water-miscible solvent, or a mixture thereof.
  • the second chamber further includes a pH adjusting agent, an inorganic salt, a saccharide, a surfactant, a chelating agent, or a combination thereof.
  • the amount of the amphiphilic block copolymer may be 0.01 to 50 parts by weight based on 1 part by weight of the cationic compound.
  • the amount of the polylactic acid salt may be 0.1 to 100 parts by weight based on 1 part by weight of the cationic compound.
  • the mixture of the amphiphilic block copolymer, the cationic compound, and the polylactate in the first chamber may be filtered one or more times with a hydrophilic filter after mixing.
  • the kit for preparing a nanoparticle composition according to the present invention includes the components for forming the drug-containing nanoparticles separated in separate chambers, unlike the already formed nanoparticles, it is not affected by the storage or transportation environment, and uses the same. Thus, the end user can successfully form nanoparticles having an effective drug delivery effect by simply mixing the components of the chamber.
  • Example 3 is a graph showing the results of evaluation of the formation of nanoparticles performed in Experimental Example 3 of the present invention using dynamic light scattering (Dynamic Light Scattering).
  • FIG. 4 is a graph showing the results of an intracellular delivery efficiency experiment of nanoparticles performed in Experimental Example 4 of the present invention.
  • Example 5 is a graph showing the results of the intracellular delivery efficiency test before and after the accelerated test performed in Experimental Example 5 of the present invention.
  • a kit for preparing a nanoparticle composition according to the present invention includes: a first chamber containing an amphiphilic block copolymer, a cationic compound, and a polylactate; and a second chamber containing an active ingredient selected from nucleic acids, polypeptides, viruses, or combinations thereof.
  • the kit of the present invention is composed of two or more chambers, and the end user can easily form nanoparticles by simply mixing the chambers.
  • the term “simple mixing” may include all acts of “mixing”, and means that no specific conditions are imposed on the act of mixing to form nanoparticles.
  • the mixing may be accomplished by various methods such as dripping, stirring (vortexing), decanting, and the like, but is not limited thereto.
  • 90% or more, 95% or more, or 99% or more of the theoretically formable amount of nanoparticles is rapidly, for example, within 1 minute, within 30 seconds, or within 15 seconds. can be formed within
  • the active ingredient in the nanoparticles formed by simple mixing by the end user can form a complex through electrostatic interaction with the cationic compound, and the complex is inside the nanoparticle structure formed by the amphiphilic block copolymer and polylactate. can be enclosed in
  • the hydrophilic portion of the amphiphilic block copolymer forms the outer wall of the nanoparticles, and the hydrophobic portion of the amphiphilic block copolymer and polylactic acid contained as separate components from the amphiphilic block copolymer
  • the salt forms the inner wall of the nanoparticles, and the complex of the active ingredient and the cationic compound may be encapsulated inside the formed nanoparticles.
  • This nanoparticle structure improves the stability of the active ingredient in blood or body fluid.
  • nucleic acid may be, for example, DNA, RNA, siRNA, shRNA, miRNA, mRNA, aptamer, antisense oligonucleotide, or a combination thereof, but is not limited thereto.
  • polypeptide includes a polypeptide sequence of an antibody or fragment thereof, a protein having activity in the body, such as a cytokine, a hormone or an analog thereof, or an antigen, an analog or a precursor thereof, and is recognized as an antigen through a series of processes in the body. It may mean a protein that can be
  • the "virus” may be an oncolytic virus, for example, adenovirus, vaccinia virus, herpes simplex virus (HSV), and may be one or more selected from the group consisting of vesicular stomatitis virus (VSV). .
  • the oncolytic virus is an adenovirus.
  • the adenovirus used in the embodiment of the present invention contains a luciferase gene, which can be confirmed through imaging.
  • the virus can express several types of therapeutic genes in the body of an individual, and is not limited to a specific molecular weight, protein, bioactivity, or therapeutic field.
  • the prophylactic virus can induce immunity in the body of a subject against a target disease.
  • a composition containing a virus for disease prevention can reduce the induction of immunity by the virus itself, can designate or expand target cells, and reduce hyperimmune response to the virus upon re-administration to obtain an effective effect by inoculating several times. have an advantage
  • the particle size of the nanoparticles may be defined as a Z-average value, for example, 800 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, or 150 nm or less. and may be 10 nm or more, 50 nm or more, or 100 nm or more.
  • the particle size of the nanoparticles as defined by the Z-average value is, for example, 10-800 nm, 10-600 nm, 10-500 nm, 10-400 nm, 10-300 nm, 10 to 200 nm or from 10 to 150 nm.
  • the “Z-average” may mean an average of hydrodynamic diameters of particle distributions measured using dynamic light scattering (DSL).
  • the nanoparticles have a monodisperse particle distribution, and the polydispersity index may be, for example, 0.01 to 0.30, 0.05 to 0.25, or 0.1 to 0.2.
  • the surface charge of the nanoparticles for example, -40 mV or more, -30 mV or more, -20 mV or more, or -10 mV or more, and also 40 mV or less, 30 mV or less, 20 mV or less, or It may be 10 mV or less.
  • the surface charge of the nanoparticles may be, for example, -40 to 40 mV, -30 to 30 mV, -20 to 20 mV, or -10 to 10 mV.
  • the surface charge may be measured in an environment close to a biological environment, for example, may be measured in 8 to 12 mM HEPES buffer (pH 7.0 to 7.5).
  • the active ingredient is a nucleic acid
  • one or more ends of the nucleic acid may be modified with one or more selected from the group consisting of cholesterol, tocopherol, and fatty acids having 10 to 24 carbon atoms.
  • the cholesterol, tocopherol and fatty acids having 10 to 24 carbon atoms include cholesterol, tocopherols and their respective analogs, derivatives, and metabolites of fatty acids.
  • the active ingredient is, based on the total weight of nanoparticles formed by the kit of the present invention, for example, 30% by weight or less, 25% by weight or less, 20% by weight or less, 15% by weight or less, 10% by weight or less, or 5 % by weight or less, and may also be 0.001 wt% or more, 0.01 wt% or more, 0.05 wt% or more, 0.1 wt% or more, 0.25 wt% or more, 0.5 wt% or more, or 1 wt% or more.
  • the active ingredient is based on the total weight of the composition, for example, 0.05 to 30% by weight, 0.1 to 25% by weight, 0.25 to 20% by weight, 0.5 to 15% by weight, 1 to 10% by weight, or 1 to 5% by weight. If the content of the active ingredient is less than the above range based on the weight of the entire composition, the amount of the carrier used compared to the drug is too large, and there may be side effects due to the carrier rather than the drug. If it exceeds the above range, the size of the nanoparticles is too large It increases, the stability of nanoparticles is reduced, and there is a fear that the loss rate during filter sterilization increases.
  • the nanoparticles may include a virus 1x10 6 to 1x10 14 VP (Virus particle), 1x10 7 to 1x10 13 VP, 1x10 8 to 1x10 12 VP, or 1x10 9 to 1x10 11 VP.
  • virus 1x10 6 to 1x10 14 VP Virus particle
  • 1x10 7 to 1x10 13 VP 1x10 8 to 1x10 12 VP
  • 1x10 9 to 1x10 11 VP 1x10 6 to 1x10 14 VP (Virus particle)
  • the cationic compound may be a cationic lipid or a cationic polymer, and more specifically, a cationic lipid.
  • the cationic lipid is N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-( 1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), N,N-dimethyl-(2,3-dioleoyloxy)propylamine (DODMA), N ,N,N-Trimethyl-(2,3-dioleoyloxy)propylamine (DOTMA), 1,2-Diacyl-3-trimethylammonium-propane (TAP), 1,2-Diacyl-3-dimethyl Ammonium-propane (DAP), 3beta-[N-(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3beta-
  • cationic lipids When using such cationic lipids, it is preferable to use less polycationic lipids having a high cation density in the molecule in order to reduce the toxicity caused by the cationic lipids, and more specifically, it is possible to represent cations in aqueous solution per molecule. It is preferable to use a cationic lipid having one functional group.
  • the cationic lipid is 3beta-[N-(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3beta[N-( N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3beta[N-(N'-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3beta[N-( Aminoethane)carbamoyl]cholesterol (AC-cholesterol), N-(1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), N,N-dimethyl-( 2,3-dioleoyloxy)propylamine (DODMA), and N,N,N-trimethyl-(2,3-
  • the cationic polymer is chitosan (chitosan), glycol chitosan (glycol chitosan), protamine (protamine), polylysine (polylysine), polyarginine (polyarginine), polyamidoamine (PAMAM), It may be selected from the group consisting of polyethyleneimine, dextran, hyaluronic acid, albumin, high molecular weight polyethyleneimine (PEI), polyamine and polyvinylamine (PVAm), and more specifically It may be at least one selected from the group consisting of polyethyleneimine (PEI), polyamine, and polyvinylamine (PVAm).
  • PEI polyethyleneimine
  • PVAm polyvinylamine
  • the cationic lipid may be a cationic lipid of Formula 1:
  • n and m are each independently 0 to 12, 2 ⁇ n + m ⁇ 12,
  • a and b are each independently 1 to 6,
  • R 1 and R 2 are each independently selected from the group consisting of saturated and unsaturated hydrocarbon groups having 11 to 25 carbon atoms.
  • n and m are each independently 1 to 9, and 2 ⁇ n+m ⁇ 10.
  • a and b may each independently be 2 to 4.
  • R 1 and R 2 are each independently, lauryl, myristyl, palmityl, stearyl, arachidyl, be Henyl (behenyl), lignoceryl (lignoceryl), cerotyl (cerotyl), myristoleyl (myristoleyl), palmitoleyl (palmitoleyl), sapienyl (sapienyl), oleyl (oleyl), linoleyl ( linoleyl), arachidonyl, eicosapentaenyl, erucyl, docosahexaenyl, and cerotyl may be selected from the group consisting of.
  • the cationic lipid is 1,6-dioleoyltriethylenetetramide (N,N'-((ethane-1,2-diylbis(azanediyl))bis(ethane-2,1-diyl) ) dioleamide), 1,8-dilinoleoyltetraethylenepentamide ((9Z,9'Z,12Z,12'Z)-N,N'-(((azanediylbis(ethane-2,1-diyl)) bis(azanediyl))bis(ethane-2,1-diyl))bis(octadeca-9,12-dienamide)), 1,4-dimyristoleoyldiethylenetriamide ((9Z,9'Z)-N ,N'-(azanediylbis(ethane-2,1-diyl))bis(tetradec-9-enamide)), 1,10
  • the content of the cationic compound in the composition formed by the kit of the present invention is, based on 1 part by weight of the active ingredient, for example, 25 parts by weight or less, 20 parts by weight or less, 18 parts by weight or less, 15 parts by weight or less, 12 parts by weight or less. It may be parts by weight or less, 10 parts by weight or less, or 8 parts by weight or less, and may also be 1 part by weight or more, 1.5 parts by weight or more, 2 parts by weight or more, 2.5 parts by weight or more, 3 parts by weight or more, or 3.5 parts by weight or more.
  • the content of the cationic compound in the composition based on 1 part by weight of the active ingredient, 1 to 25 parts by weight, 1.5 to 10 parts by weight, 2 to 15 parts by weight, 2.5 to 10 parts by weight, or 3 to 8 parts by weight.
  • the content of the cationic compound is 1 ⁇ g or more, 5 ⁇ g or more, 10 ⁇ g or more, 15 ⁇ g or more, or 18 ⁇ g or more, based on 1x10 10 VP of the virus, and 150 ⁇ g or less, 100 ⁇ g or less, 50 ⁇ g or less, or 30 ⁇ g or less, for example, 1 ⁇ g to 150 ⁇ g, 5 ⁇ g to 100 ⁇ g, 10 ⁇ g to 50 ⁇ g, 15 ⁇ g to 30 ⁇ g.
  • the content of the cationic compound in the composition is less than the above range, it may not be possible to form a stable complex with the active ingredient, and if it exceeds the above range, the size of the nanoparticles becomes too large to decrease stability and there is a risk that the loss rate during filter sterilization may increase.
  • the cationic compound and the nucleic acid are combined through an electrostatic interaction to form a complex.
  • the ratio of the charge amount of the nucleic acid (P) and the cationic compound (N) is 0.5 or more, 1 or more, 2 or more , or 3.5 or more, and may be 100 or less, 50 or less, 20 or less, for example, 0.5 to 100, 1 to 50, 2 to 20, 5 to 15, or 7 to 12.
  • the ratio (N/P) When the ratio (N/P) is less than the above range, it may be difficult to form a complex including a sufficient amount of nucleic acid, whereas when the ratio (N/P) exceeds the above range, there is a risk of causing toxicity. .
  • the N/P value may play an important role in the specific expression of the active ingredient in the spleen.
  • the amphiphilic block copolymer may be an A-B type block copolymer including a hydrophilic A block and a hydrophobic B block.
  • the A-B type block copolymer forms core-shell type polymer nanoparticles in which the hydrophobic B block forms a core (inner wall) and the hydrophilic A block forms a shell (outer wall) in an aqueous solution.
  • the hydrophilic A block may be at least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, and derivatives thereof.
  • the hydrophilic block A may be at least one selected from the group consisting of monomethoxypolyethylene glycol, monoacetoxypolyethylene glycol, polyethylene glycol, a copolymer of polyethylene and propylene glycol, and polyvinylpyrrolidone.
  • the hydrophilic A block may have a number average molecular weight of 200 to 50,000 Daltons, more specifically 1,000 to 20,000 Daltons, and even more specifically 1,000 to 5,000 Daltons.
  • amphiphilic block copolymer and polylactate are chemically bound to the end of the hydrophilic A block with a functional group capable of reaching a specific tissue or cell, a ligand, or a functional group capable of facilitating intracellular delivery. It is possible to control the distribution in the body of the polymer nanoparticle delivery system formed with the above, or to increase the efficiency of the delivery of the nanoparticle delivery system into cells.
  • the functional group or ligand may be at least one selected from the group consisting of monosaccharides, polysaccharides, vitamins, peptides, proteins, and antibodies to cell surface receptors.
  • the functional group or ligand is anisamide, vitamin B9 (folic acid), vitamin B12, vitamin A, galactose, lactose, mannose, hyaluronic acid, RGD peptide, NGR peptide, transferrin, antibody against transferrin receptor It may be one or more selected from the group consisting of.
  • the hydrophobic B block is a biocompatible biodegradable polymer, and in one embodiment, it may be one or more selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester, and polyphosphazine.
  • the hydrophobic B block is polylactide, polyglycolide, polycaprolactone, polydioxan-2-one, a copolymer of polylactide and glycolide, polylactide and polydioxan-2-one It may be at least one selected from the group consisting of a copolymer of, a copolymer of polylactide and polycaprolactone, and a copolymer of polyglycolide and polycaprolactone.
  • the hydrophobic B block may have a number average molecular weight of 50 to 50,000 Daltons, more specifically 200 to 20,000 Daltons, and even more specifically 1,000 to 5,000 Daltons.
  • the hydrophobic B block in order to increase the hydrophobicity of the hydrophobic B block to improve the stability of the nanoparticles, tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms in the hydroxy group at the end of the hydrophobic B block is chemically It may be modified by combining with .
  • the composition ratio of the hydrophilic block (A) and the hydrophobic block (B), based on the total weight of the copolymer may be in the range of 40 to 70% by weight. And, more specifically, it may be in the range of 50 to 60% by weight. If the proportion of the hydrophilic block (A) is less than 40% by weight based on the total weight of the copolymer, the solubility of the polymer in water is low and it is difficult to form nanoparticles, so that the copolymer has sufficient solubility in water to form nanoparticles.
  • the proportion of the hydrophilic block (A) is 40% by weight or more in order to have a
  • the proportion of the hydrophilic block (A) exceeds 70% by weight based on the total weight of the copolymer, the hydrophilicity is too high and the stability of the polymer nanoparticles is lowered, so it is difficult to use it as a solubilization composition of the active ingredient/cationic compound complex, In consideration of particle stability, it is preferable that the proportion of the hydrophilic block (A) is 70% by weight or less.
  • the polylactic acid salt in the composition formed by the kit of the present invention is distributed in the core (inner wall) of the nanoparticles to strengthen the hydrophobicity of the core to stabilize the nanoparticles and at the same time, the reticuloendothelial system (RES) in the body ) to effectively avoid That is, the carboxylate anion of polylactic acid binds to the cationic complex more effectively than polylactic acid and reduces the surface potential of the polymer nanoparticles. It is less captured by the endothelial system, and thus has the advantage of excellent delivery efficiency to a target site (eg, cancer cells, inflammatory cells, etc.).
  • a target site eg, cancer cells, inflammatory cells, etc.
  • the polylactic acid salt included as a component of the inner wall of the nanoparticles may have a number average molecular weight of 500 to 50,000 Daltons, and more specifically, 1,000 to 10,000 Daltons. it could be If the number average molecular weight of the polylactate is less than 500 Daltons, hydrophobicity may be too low to exist in the core (inner wall) of the nanoparticles, and if it exceeds 50,000 Daltons, there may be a problem in that the particles of the polymer nanoparticles become large.
  • the terminal opposite to the metal carboxylate (eg, sodium carboxylate) among the ends of the polylactate (eg, sodium polylactate) is hydroxy, acetoxy, benzoyloxy, decanoyloxy , may be substituted with one selected from the group consisting of palmitoyloxy and alkoxy having 1 to 2 carbon atoms.
  • A is -COO-CHZ-;
  • B is -COO-CHY-, -COO-CH 2 CH 2 CH 2 CH 2 CH 2 - or -COO-CH 2 CH 2 OCH 2 -;
  • R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl, or ethyl group;
  • Z and Y are each a hydrogen atom, or a methyl or phenyl group;
  • M is Na, K, or Li;
  • n is an integer from 1 to 30;
  • m is an integer from 0 to 20;
  • X is a methyl group
  • Y' is a hydrogen atom or a phenyl group
  • p is an integer from 0 to 25
  • q is an integer from 0 to 25, with the proviso that p+q is an integer from 5 to 25
  • R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group
  • M is Na, K, or Li
  • Z is a hydrogen atom, a methyl or phenyl group
  • W-M' is or ego
  • PAD is D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic acid, a copolymer of D,L-lactic acid and mandelic acid, D,L-lactic acid and copolymers of caprolactone and copolymers of D,L-lactic acid and 1,4-dioxan-2-one
  • R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group
  • M is independently Na, K, or Li
  • S is ego;
  • L is -NR 1 - or -0-, wherein R 1 is a hydrogen atom or C 1-10 alkyl;
  • Q is CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH 2 CH 2 CH 2 CH 3 , or CH 2 C 6 H 5 ;
  • a is an integer from 0 to 4;
  • b is an integer from 1 to 10;
  • M is Na, K, or Li;
  • PAD is D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic acid, a copolymer of D,L-lactic acid and mandelic acid, D,L-lactic acid and at least one selected from the group consisting of a copolymer of caprolactone, and a copolymer of D,L-lactic acid and 1,4-dioxan-2-one;
  • R' is -PAD-OC(O)-CH 2 CH 2 -C(O)-OM, wherein PAD is D,L-polylactic acid, D-polylactic acid, polymandelic acid, D ,L-lactic acid and glycolic acid copolymer, D,L-lactic acid and mandelic acid copolymer, D,L-lactic acid and caprolactone copolymer, D,L-lactic acid and 1,4-dioxane-2 -one is selected from the group consisting of copolymers, M is Na, K, or Li; a is an integer from 1 to 4;
  • X and X' are independently hydrogen, alkyl having 1 to 10 carbons, or aryl having 6 to 20 carbons; Y and Z are independently Na, K, or Li; m and n are independently integers from 0 to 95, provided that 5 ⁇ m + n ⁇ 100; a and b are independently integers from 1 to 6; R is -(CH 2 ) k -, divalent alkenyl having 2 to 10 carbon atoms, divalent aryl having 6 to 20 carbon atoms, or a combination thereof, where k is 0 to 10 is the integer of
  • the polylactate may be a compound of Formula 2 or Formula 3 above.
  • the kit of the present invention in order to increase the intracellular delivery efficiency of mRNA, 0.01 to 50% by weight based on the total weight of the composition formed by the kit of the present invention, more specifically 0.1 to 10 It may further comprise fusible lipids in weight percent.
  • the fusible lipid When the fusible lipid is mixed with the complex of mRNA and cationic lipid, it binds by hydrophobic interaction to form a complex of mRNA, cationic lipid and fused lipid, and the complex including the fused lipid is amphiphilic block air Encapsulated within the composite nanoparticle structure.
  • the fusible lipid may be one or a combination of two or more selected from the group consisting of phospholipids, cholesterol, and tocopherol.
  • the phospholipid may be at least one selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidic acid.
  • the phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidic acid may be combined with one or two C10-24 fatty acids.
  • the cholesterol and tocopherols include each analog, derivative, and metabolite of cholesterol and tocopherol.
  • the fusible lipids include dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, and distearoyl Ethanolamine (distearoyl phosphatidylethanolamine), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoleoyl phosphoethanolamine (1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, DPPE), dilinoleo yl phosphatidylethanolamine (dilinoleoyl phosphatidylethanolamine), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (1-palmitoyl-2-oleoyl phosphatidylethanolamine), 1,2-dipitanoyl-3-sn-phosphatidylethanolamine (1, 2-diphytanoyl-3-s
  • the fusible lipid is dioleoyl phosphatidylethanolamine (DOPE), dipalmitoleoyl phosphocholine (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, DPPC), diol as leoyl phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) and dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE) It may be one or more selected from the group consisting of.
  • DOPE dioleoyl phosphatidylethanolamine
  • DOPC dipalmitoleoyl phosphocholine
  • DOPC dipalmitoleoyl phosphocholine
  • DOPC dipalmitoleoyl phosphocholine
  • DOPC dipalmitoleoyl phosphocholine
  • DOPC dipalmitoleoyl phosphocholine
  • DOPC dipalmitoleoy
  • the content of the amphiphilic block copolymer including the hydrophilic block (A) and the hydrophobic block (B) is 1 part by weight of the cationic compound based on 0.01 to 50 parts by weight, 0.01 to 30 parts by weight, 0.01 to 15 parts by weight, 0.01 to 12 parts by weight, 0.1 to 50 parts by weight, 0.1 to 30 parts by weight, 0.1 to 15 parts by weight, 0.1 to 12 parts by weight , 0.5 to 50 parts by weight, 0.5 to 30 parts by weight, 0.5 to 15 parts by weight, 0.5 to 12 parts by weight, 1 to 50 parts by weight, 1 to 30 parts by weight, 1 to 15 parts by weight, or 1 to 12 parts by weight. have.
  • the content of the amphiphilic block copolymer may be adjusted according to the active ingredient within the above range.
  • the content of the amphiphilic block copolymer may be 3 to 7 parts by weight based on 1 part by weight of the cationic compound, and in another embodiment, when the active ingredient is a nucleic acid, The content of the amphiphilic block copolymer may be 0.7 to 7 parts by weight, 1 to 5 parts by weight, or 1 to 3 parts by weight based on 1 part by weight of the cationic compound.
  • the content of the polylactic acid salt is 0.1 to 100 parts by weight, 0.1 to 80 parts by weight, 0.1 to 100 parts by weight, based on 1 part by weight of the cationic compound. 50 parts by weight, 0.1 to 30 parts by weight, 0.1 to 10 parts by weight, 0.1 to 5 parts by weight, 0.5 to 100 parts by weight, 0.5 to 80 parts by weight, 0.5 to 50 parts by weight, 0.5 to 30 parts by weight, 1 to 100 parts by weight parts, 1 to 80 parts by weight, 1 to 50 parts by weight, 1 to 30 parts by weight, 2 to 100 parts by weight, 2 to 80 parts by weight, 2 to 50 parts by weight, or 2 to 30 parts by weight.
  • the content of the polylactic acid salt may be adjusted according to the active ingredient within the above range.
  • the content of the polylactic acid salt when the active ingredient is a virus, may be 3 to 15 parts by weight based on 1 part by weight of the cationic compound, and in another embodiment, when the active ingredient is a nucleic acid, the polylactic acid
  • the content of the salt may be 0.2 to 4 parts by weight, 1 to 4 parts by weight, or 0.2 to 0.8 parts by weight based on 1 part by weight of the cationic compound.
  • the first chamber and/or the second chamber may further include an aqueous solution, a water-miscible organic solvent, or a combination thereof.
  • aqueous solution may be used in the same sense as an aqueous solution, for example, may refer to water, sterile purified water, buffer solution, injection solution, etc., may be a buffer solution further containing an organic acid.
  • the aqueous solution may be, for example, a citric acid buffer, a PBS buffer, and the like, but is not limited thereto.
  • the "water-miscible organic solvent” may be a C1 to C4 lower alcohol, acetone, acetonitrile, a water mixture thereof, or a mixture thereof, but is not limited thereto.
  • the mixture of the amphiphilic block copolymer, the cationic compound, and the polylactic acid salt contained in the first chamber may be used as the contents of the first chamber by filtering one or more times with a hydrophilic filter after mixing.
  • the material of the hydrophilic filter is, for example, nylon, mixed cellulose ester (MCE), polyethylsulfone (PES), polyvinylidene difluoridem PVDF, cellulose acetate (CA). , polytetrafluoroethylene (PTFE), and mixtures thereof.
  • MCE mixed cellulose ester
  • PES polyethylsulfone
  • PVDF polyvinylidene difluoridem PVDF
  • CA cellulose acetate
  • PTFE polytetrafluoroethylene
  • the active ingredient may be more successfully incorporated into the nanoparticles, and the stability of the nanoparticles may be increased.
  • the second chamber may further include a stabilizer suitable for improving the stability of the active ingredient.
  • the stabilizer may further include a pH adjuster, an inorganic salt, a saccharide, a surfactant, a chelating agent, and the like, but is not limited thereto.
  • the “saccharide” may refer to monosaccharides, disaccharides, sugar alcohols that are reducing sugars thereof, polymers of single or mixed polysaccharides, and the like, and the polysaccharides may refer to three or more saccharides.
  • the monosaccharides include, for example, mannose, glucose, arabinose, fructose, galactose, and the like;
  • Examples of the disaccharide include sucrose, trehalose, maltose, lactose, cellobiose, gentiobiose, isomaltose, melibose, and the like;
  • the sugar alcohol includes mannitol, sorbitol, xylitol, erythritol, maltitol, and the like;
  • Examples of the polysaccharide include, but are not limited to, raffinose, dextran, starch, hydroxyethyl starch, cyclodextrin, cellulose, hetastarch, and oligosaccharide.
  • the “pH-adjusting agent” may be Tris, glycine, histidine, glutamate, succinate, phosphate, acetate, aspartate or a combination thereof
  • the “surfactant” may be sodium lauryl sulfate, dioctyl Sodium sulfosuccinate, dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, glycoside Deoxycholic acid sodium salt, benzalkonium chloride, Triton X-100, Triton X-114, lauromacrogol 400, polyoxyl 40 stearate, polysorbate 20, 40, 60, 65 and 80 or a combination thereof, but is not limited thereto.
  • the “chelating agent” may be citric acid, polyphenolic acid, EDTA, DTPA, EDDHA, or a combination thereof, but is not limited thereto.
  • the “inorganic salt” refers to a salt of a monovalent or divalent metal, and may be NaCl, KCl, MgCl 2 , CaCl 2 , MgSO 4 , CaSO 4 , CaCO 3 , MgCO 3 , or the like, but is not limited thereto.
  • the second chamber is 5 to 15 mM Tris, 5 to 15 mM histidine, 50 to 90 mM NaCl, 2 to 8% sucrose (w/v), 0.5 to 1.5 mM MgCl 2 , 0.005 to 0.05% (w/v) PS-80, 0.05 to 0.15 mM EDTA, and 0.1 to 1.0% ethanol (v/v), and the pH may be 7.0 to 8.0.
  • the second chamber is a PBS buffer, for example, PH 7.0 to pH 8.0, 2.0 to 3.5 mM KCl, 1.0 to 2.5 mM KH 2 PO 4 , 125 to 145 mM NaCl, and a solution comprising 7.5 to 9.5 mM Na 2 HPO 4 .
  • the "chamber” is suitable for containing a material of nanoparticles or a solvent containing the same, and includes glass, plastic, paper, pack, etc., but is not limited thereto.
  • Preparation Example 1 Preparation of first chamber composition containing dioTETA/mPEG-PLA-tocopherol (2k-1.7k)/PLANa (1.7k) and formation of nanoparticles
  • dioTETA 1,6-dioleoyl triethylenetetramide
  • mPEG-PLA-tocopherol monomethoxypolyethylene glycol-polylactide-tocopherol copolymer
  • PLANa sodium polylactate
  • A195 buffer (10 mM Tris, 10 mM histidine, 75 mM NaCl, 5% sucrose (w/v), 1 mM MgCl 2 , 0.02% (w/v) PS-80, 0.1 mM EDTA, 0.5% ethanol (v/v) v), pH 7.4) was prepared by counting VQAd CMV Luc virus (ViraQuest, Lot #: 33088) in the form dispensed to 1x10 10 VP.
  • Nanoparticles were formed by mixing the first chamber composition and the second chamber composition by vortexing for 10 to 15 seconds immediately before use.
  • MDA-MB435 cells with low CAR expression suitable for evaluating the virus delivery efficiency were prepared.
  • the nanoparticles of Examples 1 to 4 were formed by mixing the first chamber composition and the second chamber composition immediately before intracellular injection, and the cells were dispensed in an amount corresponding to 500 moi based on the virus. After additional incubation for 15 to 24 hours, luciferin was added to the cells to measure the amount of luciferase expressed.
  • a virus naked Ad; Ad
  • Preparation Example 2 Preparation of first chamber composition containing (dioTETA)/mPEG-PLA(2k-1.7k)/PLANa(1.7k) and formation of nanoparticles
  • dioTETA 20 mg in 1 ml of 20 mM sodium acetate buffer (pH 4.6), 10 mg of monomethoxypolyethylene glycol-polylactide copolymer (mPEG-PLA) (2k-1.7k) in 1 ml of water, and PLANa (1.7k)
  • mPEG-PLA monomethoxypolyethylene glycol-polylactide copolymer
  • PLANa 1.7k
  • Nanoparticles were formed by mixing the first chamber composition and the second chamber composition by vortexing for 10 to 15 seconds immediately before use.
  • Nanoparticles corresponding to Examples 6 or 8 were formed by mixing the first chamber composition and the second chamber composition immediately before intracellular injection, and were dispensed into cells in an amount corresponding to 250 ng mRNA in 96 wells. After additional incubation for 6 hours, the amount of luciferase expressed by adding luciferin to the cells was measured.
  • a commercial reagent, TransIT ® -mRNA Kit (Mirus Bio) was used. The results are shown in FIG. 4 . As a result, it was observed that the nanoparticles of the experimental example showed a similar level of intracellular delivery efficiency as the control.
  • Nanoparticles prepared with the kit have higher intracellular delivery efficiency just before administration due to the stability of the nanoparticles
  • HepG2 cells were prepared and the intracellular delivery efficiency was compared as an accelerated test.
  • Nanoparticles corresponding to Example 6 were formed by mixing the first chamber composition and the second chamber composition immediately before intracellular injection, and were dispensed into cells in an amount corresponding to 250 ng mRNA in 96 wells.
  • nanoparticles prepared in the same manner and nanoparticles prepared using L3K, which is usually used as a transfection agent, stored at 42° C. for 1 hour were used, and the same was dispensed into cells.

Abstract

The present invention relates to a kit for preparing a nanoparticle composition for drug delivery and, more specifically, to a kit for preparing a nanoparticle composition for drug delivery, which is designed to easily form a nanoparticle in which a drug is encapsulated by simply mixing an amphiphilic block copolymer, a cationic compound, a polylactic acid salt, and the drug that are the components of the kit.

Description

폴리락트산염을 포함하는 약물전달용 나노입자 조성물 제조용 키트Kit for manufacturing nanoparticle composition for drug delivery containing polylactate
본 발명은 약물전달용 나노입자 조성물을 제조하기 위한 키트에 관한 것으로, 보다 구체적으로는, 키트 구성성분인 양친성 블록 공중합체, 양이온성 화합물, 폴리락트산염 및 약물을 단순 혼합하는 것만으로 약물이 그 내부에 봉입되어 있는 나노입자를 쉽게 형성할 수 있도록 설계된 약물전달용 나노입자 조성물 제조용 키트에 관한 것이다.The present invention relates to a kit for preparing a nanoparticle composition for drug delivery, and more specifically, a drug can be prepared by simply mixing the amphiphilic block copolymer, cationic compound, polylactate, and drug, which are kit components. It relates to a kit for preparing a nanoparticle composition for drug delivery designed to easily form nanoparticles encapsulated therein.
핵산을 비롯한 음이온성 약물을 이용한 치료에 있어서, 안전하고 효율적인 약물 전달기술은 오랫동안 연구되어 왔으며, 다양한 전달체 및 전달기술이 개발되어 왔다. 전달체는 크게 아데노바이러스나 레트로바이러스 등을 이용한 바이러스성 전달체와 양이온성 지질 및 양이온성 고분자 등을 이용한 비바이러스성 전달체로 나뉜다. 바이러스성 전달체의 경우 비특이적 면역 반응 등의 위험성에 노출되어 있으며 생산 공정이 복잡하여 상용화하는 데 많은 문제점이 있는 것으로 알려져 있다. 따라서, 최근 연구 방향은 비바이러스성 전달체를 이용하여 그 단점을 개선하는 방향으로 진행되고 있다. 비바이러스성 전달체는, 바이러스성 전달체에 비하여 생체 내 안전성의 측면에서 부작용이 적고, 경제성 측면에서 생산 가격이 저렴하다는 장점을 가지고 있다.In the treatment using anionic drugs including nucleic acids, safe and efficient drug delivery technology has been studied for a long time, and various delivery systems and delivery technologies have been developed. The delivery system is largely divided into a viral delivery system using adenovirus or retrovirus, etc. and a non-viral delivery system using cationic lipids and cationic polymers. In the case of a viral carrier, it is known that there are many problems in commercialization because it is exposed to risks such as non-specific immune reaction and the production process is complicated. Therefore, the recent research direction is in the direction of improving the shortcomings by using a non-viral carrier. Compared to the viral delivery system, the non-viral delivery system has the advantages of fewer side effects in terms of in vivo safety and a low production price in terms of economic feasibility.
핵산 물질의 전달에 이용되는 비바이러스성 전달체로서 대표적인 것은 양이온성 지질을 이용한 양이온성 지질과 핵산의 복합체(lipoplex) 및 폴리양이온성(polycation) 고분자와 핵산의 복합체(polyplex)이다. 이러한 양이온성 지질 혹은 폴리양이온성 고분자는, 음이온성 약물과 정전기적 상호 작용을 통해 복합체를 형성함으로써 음이온성 약물을 안정화시키고, 세포 내 전달을 증가시킨다는 점에서 많은 연구가 진행되어 왔다(De Paula D, Bentley MV, Mahato RI, Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting, RNA 13 (2007) 431-56; Gary DJ, Puri N, Won YY, Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery, J Control release 121 (2007) 64-73).Representative non-viral carriers used for the delivery of nucleic acid substances are a complex (lipoplex) of a cationic lipid and a nucleic acid using a cationic lipid, and a complex (polyplex) of a polycationic polymer and a nucleic acid. These cationic lipids or polycationic polymers have been studied in that they stabilize the anionic drug by forming a complex through electrostatic interaction with the anionic drug and increase intracellular delivery (De Paula D) , Bentley MV, Mahato RI, Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting, RNA 13 (2007) 431-56; Gary DJ, Puri N, Won YY, Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer -based DNA delivery, J Control release 121 (2007) 64-73).
그러나 이러한 복합체가 형성하는 나노입자는 보관 환경에 따라 쉽게 안정성을 잃게 되는 경우가 잦으므로, 장기 보관에 취약하고 운송 중에 품질이 손상될 우려가 있다. 또한, 충분한 안정성을 확보하기 위해 복잡한 제조 공정을 필요로 하여 제조하기에 매우 까다로운 특징을 갖는다.However, the nanoparticles formed by these complexes often lose stability easily depending on the storage environment, so they are vulnerable to long-term storage and there is a risk that the quality may be damaged during transportation. In addition, it is very difficult to manufacture because it requires a complicated manufacturing process to ensure sufficient stability.
따라서, 보관 환경에 크게 영향을 받지 않고, 최종 소비자가 사용하기에 용이한 약물전달용 나노입자 조성물 제조용 키트의 개발이 요청되고 있다.Therefore, there is a demand for the development of a kit for preparing a nanoparticle composition for drug delivery that is not significantly affected by the storage environment and is easy for end-users to use.
본 발명의 목적은 키트 구성성분들을 단순 혼합하는 것만으로 약물이 함유된 나노입자를 쉽게 형성할 수 있어 최종 소비자가 사용하기에 용이하고, 약물-함유 나노입자를 사용 직전에 쉽게 형성할 수 있으므로 그 보관 또는 운송 환경에 따른 영향 없이 약물을 체내에 효과적으로 전달할 수 있는, 약물전달용 나노입자 조성물 제조용 키트를 제공하는 것이다.It is an object of the present invention to easily form drug-containing nanoparticles by simply mixing the kit components so that it is easy for the end consumer to use, and drug-containing nanoparticles can be easily formed immediately before use. An object of the present invention is to provide a kit for preparing a nanoparticle composition for drug delivery, which can effectively deliver a drug into the body without the influence of storage or transportation environment.
상기 과제를 해결하고자 본 발명은, 양친성 블록 공중합체, 양이온성 화합물 및 폴리락트산염을 포함하는 제1 챔버; 및 핵산, 폴리펩티드, 바이러스 또는 이들의 조합으로부터 선택된 유효성분을 포함하는 제2 챔버;를 포함하는, 나노입자 조성물 제조용 키트를 제공한다.In order to solve the above problems, the present invention provides a first chamber containing an amphiphilic block copolymer, a cationic compound, and a polylactate; and a second chamber containing an active ingredient selected from a nucleic acid, a polypeptide, a virus, or a combination thereof; provides a kit for preparing a nanoparticle composition comprising a.
일 구체예에서, 상기 키트는 세포 내 유효성분을 전달하는 나노입자를 형성하기 위한 것이다.In one embodiment, the kit is for forming nanoparticles that deliver an intracellular active ingredient.
일 구체예에서, 상기 제1 챔버 및 제2 챔버로 이루어진 군 중에서 선택된 하나 이상은 추가의 용매를 더 포함한다.In one embodiment, at least one selected from the group consisting of the first chamber and the second chamber further includes an additional solvent.
일 구체예에서, 상기 용매는 수성 용매, 수혼화성 용매 또는 이의 혼합물이다.In one embodiment, the solvent is an aqueous solvent, a water-miscible solvent, or a mixture thereof.
일 구체예에서, 상기 제2 챔버는 pH 조절제, 무기염, 당류, 계면활성제, 킬레이트제 또는 이들의 조합을 더 포함한다.In one embodiment, the second chamber further includes a pH adjusting agent, an inorganic salt, a saccharide, a surfactant, a chelating agent, or a combination thereof.
일 구체예에서, 상기 양친성 블록 공중합체의 양은 상기 양이온성 화합물 1 중량부를 기준으로 0.01 내지 50 중량부일 수 있다.In one embodiment, the amount of the amphiphilic block copolymer may be 0.01 to 50 parts by weight based on 1 part by weight of the cationic compound.
일 구체예에서, 상기 폴리락트산염의 양은 상기 양이온성 화합물 1 중량부를 기준으로 0.1 내지 100 중량부일 수 있다.In one embodiment, the amount of the polylactic acid salt may be 0.1 to 100 parts by weight based on 1 part by weight of the cationic compound.
일 구체예에서, 상기 제1 챔버에서 상기 양친성 블록 공중합체, 양이온성 화합물 및 폴리락트산염의 혼합물은, 혼합 후 친수성 필터로 1회 이상 여과된 것일 수 있다.In one embodiment, the mixture of the amphiphilic block copolymer, the cationic compound, and the polylactate in the first chamber may be filtered one or more times with a hydrophilic filter after mixing.
본 발명에 따른 나노입자 조성물 제조용 키트는, 약물-함유 나노입자를 형성하기 위한 구성성분들을 별도의 챔버들 내에 분리시켜 포함하므로, 이미 형성된 나노입자와 달리 보관 또는 운송 환경에 영향받지 않으며, 이를 사용하면 최종 사용자가 챔버의 성분들을 단순 혼합하는 것 만으로도 유효한 약물 전달 효과를 갖는 나노입자를 성공적으로 형성할 수 있다.Since the kit for preparing a nanoparticle composition according to the present invention includes the components for forming the drug-containing nanoparticles separated in separate chambers, unlike the already formed nanoparticles, it is not affected by the storage or transportation environment, and uses the same. Thus, the end user can successfully form nanoparticles having an effective drug delivery effect by simply mixing the components of the chamber.
도 1은 본 발명의 실험예 2에서 수행된 나노입자의 세포 내 전달 효율 실험 결과를 보여주는 그래프이다.1 is a graph showing the results of an intracellular delivery efficiency experiment of nanoparticles performed in Experimental Example 2 of the present invention.
도 2는 본 발명의 실험예 3에서 수행된 나노 입자의 형성을 아가로오스 젤 분석을 통해 확인한 결과를 보여주는 이미지이다.2 is an image showing the result of confirming the formation of nanoparticles performed in Experimental Example 3 of the present invention through agarose gel analysis.
도 3은 본 발명의 실험예 3에서 수행된 나노입자의 형성을 동적광산란 (Dynamic Light Scattering)을 이용하여 평가한 결과를 보여주는 그래프이다.3 is a graph showing the results of evaluation of the formation of nanoparticles performed in Experimental Example 3 of the present invention using dynamic light scattering (Dynamic Light Scattering).
도 4는 본 발명의 실험예 4에서 수행된 나노입자의 세포 내 전달 효율 실험 결과를 보여주는 그래프이다.4 is a graph showing the results of an intracellular delivery efficiency experiment of nanoparticles performed in Experimental Example 4 of the present invention.
도 5는 본 발명의 실험예 5에서 수행된 가속시험 전후 세포 내 전달 효율 실험 결과를 보여주는 그래프이다.5 is a graph showing the results of the intracellular delivery efficiency test before and after the accelerated test performed in Experimental Example 5 of the present invention.
이하, 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명에 따른 나노입자 조성물 제조용 키트는, 양친성 블록 공중합체, 양이온성 화합물 및 폴리락트산염을 포함하는 제1 챔버; 및 핵산, 폴리펩티드, 바이러스 또는 이들의 조합으로부터 선택된 유효성분을 포함하는 제2 챔버;를 포함한다.A kit for preparing a nanoparticle composition according to the present invention includes: a first chamber containing an amphiphilic block copolymer, a cationic compound, and a polylactate; and a second chamber containing an active ingredient selected from nucleic acids, polypeptides, viruses, or combinations thereof.
본 발명의 키트는, 두 개 이상의 챔버로 구성되어 있고, 최종 사용자는 상기 챔버들을 단순 혼합함으로써 나노입자를 쉽게 형성할 수 있다. 용어 “단순 혼합”은 “혼합”하는 행위를 모두 포함할 수 있고, 나노입자를 형성하기 위해 혼합하는 행위에 특정한 조건이 부여되지 않는 것을 의미한다. 상기 혼합은, 점적, 교반(vortexing), 디켄팅 등 다양한 방식에 의해 이루어질 수 있고, 이에 제한되지 않는다. 일 구체예에 따르면, 본 발명의 키트 사용시, 이론적으로 형성 가능한 양의 90% 이상, 95 % 이상, 또는 99% 이상의 나노입자가 신속히, 예를 들어, 1분 이내, 30초 이내, 또는 15 초 이내에 형성될 수 있다.The kit of the present invention is composed of two or more chambers, and the end user can easily form nanoparticles by simply mixing the chambers. The term “simple mixing” may include all acts of “mixing”, and means that no specific conditions are imposed on the act of mixing to form nanoparticles. The mixing may be accomplished by various methods such as dripping, stirring (vortexing), decanting, and the like, but is not limited thereto. According to one embodiment, when using the kit of the present invention, 90% or more, 95% or more, or 99% or more of the theoretically formable amount of nanoparticles is rapidly, for example, within 1 minute, within 30 seconds, or within 15 seconds. can be formed within
최종 사용자가 단순 혼합하여 형성하는 나노입자 내의 유효성분은 양이온성 화합물과 정전기적 상호작용을 통해 복합체를 형성할 수 있고, 상기 복합체는 양친성 블록 공중합체와 폴리락트산염에 의해 형성된 나노입자 구조 내부에 봉입될 수 있다. The active ingredient in the nanoparticles formed by simple mixing by the end user can form a complex through electrostatic interaction with the cationic compound, and the complex is inside the nanoparticle structure formed by the amphiphilic block copolymer and polylactate. can be enclosed in
상기 나노입자에서, 수성 환경에서는 양친성 블록 공중합체의 친수성 부분이 나노입자의 외벽을 형성하고, 양친성 블록 공중합체의 소수성 부분과 상기 양친성 블록 공중합체와는 별도의 성분으로 함유된 폴리락트산염이 나노입자의 내벽을 형성하며, 그 형성된 나노입자의 내부에 유효성분과 양이온성 화합물의 복합체가 봉입될 수 있다. 이러한 나노입자 구조는, 혈중 또는 체액 내에서 유효성분의 안정성을 향상시킨다.In the above nanoparticles, in an aqueous environment, the hydrophilic portion of the amphiphilic block copolymer forms the outer wall of the nanoparticles, and the hydrophobic portion of the amphiphilic block copolymer and polylactic acid contained as separate components from the amphiphilic block copolymer The salt forms the inner wall of the nanoparticles, and the complex of the active ingredient and the cationic compound may be encapsulated inside the formed nanoparticles. This nanoparticle structure improves the stability of the active ingredient in blood or body fluid.
상기 “핵산”은, 예를 들어, DNA, RNA, siRNA, shRNA, miRNA, mRNA, 앱타머, 안티센스 올리고뉴클레오티드, 또는 이들의 조합일 수 있으나, 이에 제한되지 않는다. The “nucleic acid” may be, for example, DNA, RNA, siRNA, shRNA, miRNA, mRNA, aptamer, antisense oligonucleotide, or a combination thereof, but is not limited thereto.
상기 “폴리펩티드”는 항체 또는 이의 절편, 시토킨, 호르몬 또는 그 유사체와 같은 체내에 활성을 갖는 단백질, 또는 항원, 이의 유사체 또는 전구체의 폴리펩티드 서열을 포함하여, 체내에서 일련의 과정을 통해 항원으로 인식될 수 있는 단백질을 의미할 수 있다.The "polypeptide" includes a polypeptide sequence of an antibody or fragment thereof, a protein having activity in the body, such as a cytokine, a hormone or an analog thereof, or an antigen, an analog or a precursor thereof, and is recognized as an antigen through a series of processes in the body. It may mean a protein that can be
상기 “바이러스”는 암살상 바이러스(oncolytic virus)일 수 있고, 예를 들어, 아데노바이러스, 백시니아바이러스, 단순헤르페스바이러스(HSV) 및 수포성구내염바이러스(VSV)로 구성된 그룹으로부터 선택된 하나 이상일 수 있다. 일 구체예에서, 암살상 바이러스는 아데노바이러스이다. 본 발명의 구체예에서 사용된 아데노바이러스는 루시퍼라아제 유전자를 포함하고, 이것은 이미징을 통해 확인할 수 있다. The "virus" may be an oncolytic virus, for example, adenovirus, vaccinia virus, herpes simplex virus (HSV), and may be one or more selected from the group consisting of vesicular stomatitis virus (VSV). . In one embodiment, the oncolytic virus is an adenovirus. The adenovirus used in the embodiment of the present invention contains a luciferase gene, which can be confirmed through imaging.
상기 바이러스는 개체의 체내에 여러 종류의 치료 유전자를 발현할 수 있으며, 특정 분자량, 단백질, 생활성 또는 치료 분야에 제한되지 않는다. 예방용 바이러스는 표적 질병에 대해 개체의 체내에 면역을 유발할 수 있다. 질병 예방용 바이러스를 포함하는 조성물은 바이러스 자체에 의한 면역 유발을 감소시키고, 표적 세포를 지정 또는 확장 가능하며, 재투여시 바이러스에 대한 과면역 반응을 감소시켜 수 회 접종하여 유효한 효과를 얻을 수 있는 장점을 갖는다.The virus can express several types of therapeutic genes in the body of an individual, and is not limited to a specific molecular weight, protein, bioactivity, or therapeutic field. The prophylactic virus can induce immunity in the body of a subject against a target disease. A composition containing a virus for disease prevention can reduce the induction of immunity by the virus itself, can designate or expand target cells, and reduce hyperimmune response to the virus upon re-administration to obtain an effective effect by inoculating several times. have an advantage
일 예에서, 상기 나노입자의 입자 크기는 Z-평균 값으로 정의될 수 있고, 예컨대, 800 nm 이하, 600 nm 이하, 500 nm 이하, 400 nm 이하, 300 nm 이하, 200 nm 이하 또는 150 nm 이하일 수 있으며, 또한 10 nm 이상, 50 nm 이상, 또는 100 nm 이상일 수 있다. 일 구체예에서, Z-평균 값으로 정의된 상기 나노입자의 입자 크기는, 예를 들어, 10 내지 800 nm, 10 내지 600 nm, 10 내지 500 nm, 10 내지 400 nm, 10 내지 300 nm, 10 내지 200 nm 또는 10 내지 150 nm일 수 있다. In one example, the particle size of the nanoparticles may be defined as a Z-average value, for example, 800 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, or 150 nm or less. and may be 10 nm or more, 50 nm or more, or 100 nm or more. In one embodiment, the particle size of the nanoparticles as defined by the Z-average value is, for example, 10-800 nm, 10-600 nm, 10-500 nm, 10-400 nm, 10-300 nm, 10 to 200 nm or from 10 to 150 nm.
상기 “Z-평균”은 동적빛산란(Dynamic light scattering, DSL)을 이용하여 측정한 입자 분포의 유체학적 지름(hydrodynamic diameter)의 평균을 의미할 수 있다. 상기 나노입자는 단순분산 입자 분포(monodisperse particle distribution)를 갖는 것으로서, 그 다분산도 지수(polydispersity index)는, 예컨대, 0.01 내지 0.30, 0.05 내지 0.25, 또는 0.1 내지 0.2일 수 있다.The “Z-average” may mean an average of hydrodynamic diameters of particle distributions measured using dynamic light scattering (DSL). The nanoparticles have a monodisperse particle distribution, and the polydispersity index may be, for example, 0.01 to 0.30, 0.05 to 0.25, or 0.1 to 0.2.
또한, 일 예에서, 상기 나노입자의 표면 전하는, 예컨대, -40 mV 이상, -30 mV 이상, -20 mV 이상 또는 -10 mV 이상일 수 있고, 또한 40 mV 이하, 30 mV 이하, 20 mV 이하 또는 10 mV 이하일 수 있다. 일 구체예에서, 상기 나노입자의 표면 전하는, 예를 들어, -40 내지 40 mV, -30 내지 30 mV, -20 내지 20 mV 또는 -10 내지 10 mV일 수 있다. 상기 표면 전하는 생물학적 환경에 가까운 환경에서 측정된 것일 수 있고, 예를 들어, 8 내지 12 mM HEPES 완충액(pH 7.0 내지 7.5)에서 측정될 수 있다.Further, in one example, the surface charge of the nanoparticles, for example, -40 mV or more, -30 mV or more, -20 mV or more, or -10 mV or more, and also 40 mV or less, 30 mV or less, 20 mV or less, or It may be 10 mV or less. In one embodiment, the surface charge of the nanoparticles may be, for example, -40 to 40 mV, -30 to 30 mV, -20 to 20 mV, or -10 to 10 mV. The surface charge may be measured in an environment close to a biological environment, for example, may be measured in 8 to 12 mM HEPES buffer (pH 7.0 to 7.5).
나노입자의 입자 크기 및 표면 전하가 상기 수준을 유지할 경우, 나노입자 구조의 안정성 및 구성성분들의 함량과 체내에서 흡수도 및 약제학적 조성물로서 멸균의 편의성 측면에서 바람직하다. 예를 들어, 상기 유효성분이 핵산인 경우, 상기 핵산의 하나 이상의 말단은 콜레스테롤, 토코페롤 및 탄소수 10 내지 24개의 지방산으로 구성된 군으로부터 선택되는 하나 이상으로 수식될 수 있다. 상기 콜레스테롤, 토코페롤 및 탄소수 10 내지 24개의 지방산에는 콜레스테롤, 토코페롤 및 지방산의 각 유사체, 유도체, 및 대사체가 포함된다.When the particle size and surface charge of the nanoparticles are maintained at the above levels, it is preferable in terms of stability of the nanoparticle structure, content of components, absorption in the body and convenience of sterilization as a pharmaceutical composition. For example, when the active ingredient is a nucleic acid, one or more ends of the nucleic acid may be modified with one or more selected from the group consisting of cholesterol, tocopherol, and fatty acids having 10 to 24 carbon atoms. The cholesterol, tocopherol and fatty acids having 10 to 24 carbon atoms include cholesterol, tocopherols and their respective analogs, derivatives, and metabolites of fatty acids.
상기 유효성분은, 본 발명의 키트에 의하여 형성되는 전체 나노입자 중량을 기준으로, 예컨대, 30 중량% 이하, 25 중량% 이하, 20 중량% 이하, 15 중량% 이하, 10 중량% 이하, 또는 5 중량% 이하일 수 있고, 또한 0.001 중량% 이상, 0.01 중량% 이상, 0.05 중량% 이상, 0.1 중량% 이상, 0.25 중량% 이상, 0.5 중량% 이상 또는 1 중량% 이상일 수 있다. 일 구체예에서, 상기 유효성분은 전체 조성물 중량을 기준으로, 예를 들어, 0.05 내지 30 중량%, 0.1 내지 25 중량%, 0.25 내지 20 중량%, 0.5 내지 15 중량%, 1 내지 10 중량%, 또는 1 내지 5 중량%일 수 있다. 유효성분의 함량이 전체 조성물의 중량을 기준으로 상기 범위보다 적으면 약물에 비하여 사용되는 전달체의 양이 너무 많아서 오히려 전달체에 의한 부작용이 있을 수 있고, 상기 범위를 초과하면, 나노입자의 크기가 너무 커져 나노입자의 안정성이 저하되고 필터 멸균시 손실율이 커질 우려가 있다. 상기 유효성분이 바이러스인 경우, 상기 나노입자는 바이러스 1x106 내지 1x1014VP(Virus particle), 1x107 내지 1x1013VP, 1x108 내지 1x1012VP, 또는 1x109 내지 1x1011VP를 포함할 수 있다.The active ingredient is, based on the total weight of nanoparticles formed by the kit of the present invention, for example, 30% by weight or less, 25% by weight or less, 20% by weight or less, 15% by weight or less, 10% by weight or less, or 5 % by weight or less, and may also be 0.001 wt% or more, 0.01 wt% or more, 0.05 wt% or more, 0.1 wt% or more, 0.25 wt% or more, 0.5 wt% or more, or 1 wt% or more. In one embodiment, the active ingredient is based on the total weight of the composition, for example, 0.05 to 30% by weight, 0.1 to 25% by weight, 0.25 to 20% by weight, 0.5 to 15% by weight, 1 to 10% by weight, or 1 to 5% by weight. If the content of the active ingredient is less than the above range based on the weight of the entire composition, the amount of the carrier used compared to the drug is too large, and there may be side effects due to the carrier rather than the drug. If it exceeds the above range, the size of the nanoparticles is too large It increases, the stability of nanoparticles is reduced, and there is a fear that the loss rate during filter sterilization increases. When the active ingredient is a virus, the nanoparticles may include a virus 1x10 6 to 1x10 14 VP (Virus particle), 1x10 7 to 1x10 13 VP, 1x10 8 to 1x10 12 VP, or 1x10 9 to 1x10 11 VP.
구체적인 일 태양에서, 상기 양이온성 화합물은 양이온성 지질 또는 양이온성 고분자 종류일 수 있으며, 보다 구체적으로는 양이온성 지질일 수 있다.In a specific embodiment, the cationic compound may be a cationic lipid or a cationic polymer, and more specifically, a cationic lipid.
일 구체예에서, 상기 양이온성 지질은 N,N-디올레일-N,N-디메틸암모늄클로라이드(DODAC), N,N-디스테아릴-N,N-디메틸암모늄브로마이드(DDAB), N-(1-(2,3-디올레오일옥시)프로필-N,N,N-트리메틸암모늄클로라이드(DOTAP), N,N-디메틸-(2,3-디올레오일옥시)프로필아민(DODMA), N,N,N-트리메틸-(2,3-디올레오일옥시)프로필아민(DOTMA), 1,2-디아실-3-트리메틸암모늄-프로판(TAP), 1,2-디아실-3-디메틸암모늄-프로판(DAP), 3베타-[N-(N',N',N'-트리메틸아미노에탄)카바모일]콜레스테롤(TC-콜레스테롤), 3베타-[N-(N',N'-디메틸아미노에탄)카바모일]콜레스테롤(DC-콜레스테롤), 3베타-[N-(N'-모노메틸아미노에탄)카바모일]콜레스테롤(MC-콜레스테롤), 3베타-[N-(아미노에탄)카바모일]콜레스테롤(AC-콜레스테롤), 콜레스테릴옥시프로판-1-아민(COPA), N-(N'-아미노에탄)카바모일프로파노익 토코페롤(AC-토코페롤) 및 N-(N'-메틸아미노에탄)카바모일프로파노익 토코페롤(MC-토코페롤)로 구성된 군으로부터 선택된 하나 또는 둘 이상의 조합일 수 있다. In one embodiment, the cationic lipid is N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-( 1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), N,N-dimethyl-(2,3-dioleoyloxy)propylamine (DODMA), N ,N,N-Trimethyl-(2,3-dioleoyloxy)propylamine (DOTMA), 1,2-Diacyl-3-trimethylammonium-propane (TAP), 1,2-Diacyl-3-dimethyl Ammonium-propane (DAP), 3beta-[N-(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3beta-[N-(N',N'-) Dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3beta-[N-(N'-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3beta-[N-(aminoethane)carba Moyl]cholesterol (AC-cholesterol), cholesteryloxypropan-1-amine (COPA), N-(N'-aminoethane)carbamoylpropanoic tocopherol (AC-tocopherol) and N-(N'-methyl) It may be one or a combination of two or more selected from the group consisting of aminoethane) carbamoylpropanoic tocopherol (MC-tocopherol).
이러한 양이온성 지질을 사용하는 경우, 양이온성 지질로부터 유발되는 독성을 감소시키기 위하여 분자 내의 양이온 밀도가 높은 폴리양이온성 지질을 적게 사용하는 것이 바람직하고, 보다 구체적으로는 분자당 수용액 상에서 양이온을 나타낼 수 있는 작용기가 하나인 양이온성 지질을 사용하는 것이 바람직하다. When using such cationic lipids, it is preferable to use less polycationic lipids having a high cation density in the molecule in order to reduce the toxicity caused by the cationic lipids, and more specifically, it is possible to represent cations in aqueous solution per molecule. It is preferable to use a cationic lipid having one functional group.
이에 따라, 보다 바람직한 일 태양에서, 상기 양이온성 지질은 3베타-[N-(N',N',N'-트리메틸아미노에탄)카바모일]콜레스테롤(TC-콜레스테롤), 3베타[N-(N',N'- 디메틸아미노에탄)카바모일]콜레스테롤(DC-콜레스테롤), 3베타[N-(N'- 모노메틸아미노에탄)카바모일]콜레스테롤(MC-콜레스테롤), 3베타[N-(아미노에탄)카바모일]콜레스테롤(AC-콜레스테롤), N-(1-(2,3-디올레오일옥시) 프로필-N,N,N-트리메틸암모늄클로라이드(DOTAP), N,N-디메틸-(2,3-디올레오일옥시)프로필아민(DODMA), 및 N,N,N-트리메틸-(2,3-디올레오일옥시)프로필아민(DOTMA)으로 이루어진 군에서 선택된 1종 이상의 것일 수 있다. Accordingly, in a more preferred embodiment, the cationic lipid is 3beta-[N-(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3beta[N-( N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3beta[N-(N'-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3beta[N-( Aminoethane)carbamoyl]cholesterol (AC-cholesterol), N-(1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), N,N-dimethyl-( 2,3-dioleoyloxy)propylamine (DODMA), and N,N,N-trimethyl-(2,3-dioleoyloxy)propylamine (DOTMA) may be at least one selected from the group consisting of .
한편, 일 구체예에서, 상기 양이온성 고분자는 키토산(chitosan), 글라이콜 키토산(glycol chitosan), 프로타민(protamine), 폴리라이신(polylysine), 폴리아르기닌(polyarginine), 폴리아미도아민(PAMAM), 폴리에틸렌이민(polyethylenimine), 덱스트란(dextran), 히알루론산(hyaluronic acid), 알부민(albumin), 고분자폴리에틸렌이민(PEI), 폴리아민 및 폴리비닐아민(PVAm)으로 구성된 군으로부터 선택될 수 있으며, 보다 구체적으로는 폴리에틸렌이민(PEI), 폴리아민 및 폴리비닐아민(PVAm)으로 이루어진 군으로부터 선택되는 1종 이상의 것일 수 있다.On the other hand, in one embodiment, the cationic polymer is chitosan (chitosan), glycol chitosan (glycol chitosan), protamine (protamine), polylysine (polylysine), polyarginine (polyarginine), polyamidoamine (PAMAM), It may be selected from the group consisting of polyethyleneimine, dextran, hyaluronic acid, albumin, high molecular weight polyethyleneimine (PEI), polyamine and polyvinylamine (PVAm), and more specifically It may be at least one selected from the group consisting of polyethyleneimine (PEI), polyamine, and polyvinylamine (PVAm).
구체적인 일 태양에서, 상기 양이온성 지질은 하기 화학식 1의 양이온성 지질일 수 있다:In a specific embodiment, the cationic lipid may be a cationic lipid of Formula 1:
[화학식 1][Formula 1]
Figure PCTKR2020018480-appb-I000001
Figure PCTKR2020018480-appb-I000001
상기 식에서,In the above formula,
n 및 m은 각각 독립적으로 0 내지 12이되, 2 ≤ n + m ≤ 12이고,n and m are each independently 0 to 12, 2 ≤ n + m ≤ 12,
a 및 b는 각각 독립적으로 1 내지 6이며, a and b are each independently 1 to 6,
R1 및 R2는 각각 독립적으로 탄소수 11 내지 25개의 포화 및 불포화 탄화수소기로 이루어진 군에서 선택된 것이다.R 1 and R 2 are each independently selected from the group consisting of saturated and unsaturated hydrocarbon groups having 11 to 25 carbon atoms.
보다 구체적으로, 상기 화학식 1에서, n 및 m은 각각 독립적으로 1 내지 9이며, 2 ≤ n+m ≤ 10일 수 있다.More specifically, in Formula 1, n and m are each independently 1 to 9, and 2 ≤ n+m ≤ 10.
보다 구체적으로, 상기 화학식 1에서, a 및 b는 각각 독립적으로 2 내지 4일 수 있다.More specifically, in Formula 1, a and b may each independently be 2 to 4.
보다 구체적으로, 상기 화학식 1에서, R1 및 R2는 각각 독립적으로, 라우릴(lauryl), 미리스틸(myristyl), 팔미틸(palmityl), 스테아릴(stearyl), 아라키딜(arachidyl), 베헨닐(behenyl), 리그노세릴(lignoceryl), 세로틸(cerotyl), 미리스트올레일(myristoleyl), 팔미트올레일(palmitoleyl), 사피에닐(sapienyl), 올레일(oleyl), 리놀레일(linoleyl), 아라키도닐(arachidonyl), 에이코사펜타에닐(eicosapentaenyl), 에루실(erucyl), 도코사헥사에닐(docosahexaenyl), 및 세로틸(cerotyl)로 이루어진 군에서 선택된 것일 수 있다.More specifically, in Formula 1, R 1 and R 2 are each independently, lauryl, myristyl, palmityl, stearyl, arachidyl, be Henyl (behenyl), lignoceryl (lignoceryl), cerotyl (cerotyl), myristoleyl (myristoleyl), palmitoleyl (palmitoleyl), sapienyl (sapienyl), oleyl (oleyl), linoleyl ( linoleyl), arachidonyl, eicosapentaenyl, erucyl, docosahexaenyl, and cerotyl may be selected from the group consisting of.
일 구체예에서, 상기 양이온성 지질은 1,6-디올레오일트리에틸렌테트라마이드(N,N'-((ethane-1,2-diylbis(azanediyl))bis(ethane-2,1-diyl))dioleamide), 1,8-디리놀레오일테트라에틸렌펜타마이드((9Z,9'Z,12Z,12'Z)-N,N'-(((azanediylbis(ethane-2,1-diyl))bis(azanediyl))bis(ethane-2,1-diyl))bis(octadeca-9,12-dienamide)), 1,4-디미리스톨레오일디에틸렌트리아마이드((9Z,9'Z)-N,N'-(azanediylbis(ethane-2,1-diyl))bis(tetradec-9-enamide)), 1,10-디스테아로일펜타에틸렌헥사마이드(N,N'-(3,6,9,12-tetraazatetradecane-1,14-diyl)distearamide) 및 1,10-디올레오일펜타에틸렌헥사마이드(N,N'-(3,6,9,12-tetraazatetradecane-1,14-diyl)dioleamide)로 구성된 그룹으로부터 선택되는 하나 이상일 수 있다.In one embodiment, the cationic lipid is 1,6-dioleoyltriethylenetetramide (N,N'-((ethane-1,2-diylbis(azanediyl))bis(ethane-2,1-diyl) ) dioleamide), 1,8-dilinoleoyltetraethylenepentamide ((9Z,9'Z,12Z,12'Z)-N,N'-(((azanediylbis(ethane-2,1-diyl)) bis(azanediyl))bis(ethane-2,1-diyl))bis(octadeca-9,12-dienamide)), 1,4-dimyristoleoyldiethylenetriamide ((9Z,9'Z)-N ,N'-(azanediylbis(ethane-2,1-diyl))bis(tetradec-9-enamide)), 1,10-distearoylpentaethylenehexaamide (N,N'-(3,6,9) ,12-tetraazatetradecane-1,14-diyl)distearamide) and 1,10-dioleoylpentaethylenehexamide (N,N'-(3,6,9,12-tetraazatetradecane-1,14-diyl)dioleamide) It may be one or more selected from the group consisting of.
본 발명의 키트에 의하여 형성되는 조성물 내의 상기 양이온성 화합물의 함량은, 유효성분 1 중량부를 기준으로, 예컨대, 25 중량부 이하, 20 중량부 이하, 18 중량부 이하, 15 중량부 이하, 12 중량부 이하, 10 중량부 이하, 또는 8 중량부 이하일 수 있고, 또한 1 중량부 이상, 1.5 중량부 이상, 2 중량부 이상, 2.5 중량부 이상, 3 중량부 이상, 또는 3.5 중량부 이상일 수 있다. 일 구체예에서, 조성물 내의 상기 양이온성 화합물의 함량은, 유효성분 1 중량부를 기준으로, 1 내지 25 중량부, 1.5 내지 10 중량부, 2 내지 15 중량부, 2.5 내지 10 중량부, 또는 3 내지 8 중량부일 수 있다. 한편, 유효성분이 바이러스, 보다 구체적으로 아데노바이러스인 경우, 바이러스 1x1010VP를 기준으로 상기 양이온성 화합물의 함량은 1㎍ 이상, 5㎍ 이상, 10㎍ 이상, 15㎍ 이상 또는 18㎍ 이상이면서, 150㎍ 이하, 100㎍ 이하, 50㎍ 이하, 또는 30㎍ 이하로서, 예를 들어, 1 ㎍ 내지 150 ㎍, 5 ㎍ 내지 100 ㎍, 10 ㎍ 내지 50 ㎍, 15 ㎍ 내지 30 ㎍일 수 있다. 조성물 내의 상기 양이온성 화합물의 함량이 상기 범위 미만이면 유효성분과 안정한 복합체를 형성하지 못할 수 있고, 상기 범위를 초과하면 나노입자의 크기가 너무 커져 안정성이 저하되고 필터 멸균시 손실율이 커질 우려가 있다. The content of the cationic compound in the composition formed by the kit of the present invention is, based on 1 part by weight of the active ingredient, for example, 25 parts by weight or less, 20 parts by weight or less, 18 parts by weight or less, 15 parts by weight or less, 12 parts by weight or less. It may be parts by weight or less, 10 parts by weight or less, or 8 parts by weight or less, and may also be 1 part by weight or more, 1.5 parts by weight or more, 2 parts by weight or more, 2.5 parts by weight or more, 3 parts by weight or more, or 3.5 parts by weight or more. In one embodiment, the content of the cationic compound in the composition, based on 1 part by weight of the active ingredient, 1 to 25 parts by weight, 1.5 to 10 parts by weight, 2 to 15 parts by weight, 2.5 to 10 parts by weight, or 3 to 8 parts by weight. On the other hand, when the active ingredient is a virus, more specifically, adenovirus, the content of the cationic compound is 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, or 18 μg or more, based on 1x10 10 VP of the virus, and 150 μg or less, 100 μg or less, 50 μg or less, or 30 μg or less, for example, 1 μg to 150 μg, 5 μg to 100 μg, 10 μg to 50 μg, 15 μg to 30 μg. If the content of the cationic compound in the composition is less than the above range, it may not be possible to form a stable complex with the active ingredient, and if it exceeds the above range, the size of the nanoparticles becomes too large to decrease stability and there is a risk that the loss rate during filter sterilization may increase.
상기 유효성분이 핵산인 경우, 상기 양이온성 화합물과 핵산은 정전기적 상호작용을 통해 결합하여 복합체를 형성한다. 일 구체예에서, 상기 핵산(P)과 양이온성 화합물(N)의 전하량의 비율(N/P; 핵산의 음이온 전하에 대한 양이온성 화합물의 양이온 전하 비율)은, 0.5 이상, 1 이상, 2 이상, 또는 3.5 이상일 수 있고, 또한 100 이하, 50 이하, 20 이하일 수 있으며, 예를 들어, 0.5 내지 100, 1 내지 50, 2 내지 20, 5 내지 15, 또는 7 내지 12일 수 있다. 상기 비율(N/P)이 상기 범위 미만인 경우에는 충분한 양의 핵산을 포함하는 복합체를 형성하기 어려울 수 있는 반면, 상기 비율(N/P)이 상기 범위를 초과할 시에는 독성을 유발할 우려가 있다. 또한, N/P 값은 유효성분이 비장에서 특이적으로 발현하는데 중요하게 작용할 수 있다.When the active ingredient is a nucleic acid, the cationic compound and the nucleic acid are combined through an electrostatic interaction to form a complex. In one embodiment, the ratio of the charge amount of the nucleic acid (P) and the cationic compound (N) (N/P; ratio of the cationic charge of the cationic compound to the anionic charge of the nucleic acid) is 0.5 or more, 1 or more, 2 or more , or 3.5 or more, and may be 100 or less, 50 or less, 20 or less, for example, 0.5 to 100, 1 to 50, 2 to 20, 5 to 15, or 7 to 12. When the ratio (N/P) is less than the above range, it may be difficult to form a complex including a sufficient amount of nucleic acid, whereas when the ratio (N/P) exceeds the above range, there is a risk of causing toxicity. . In addition, the N/P value may play an important role in the specific expression of the active ingredient in the spleen.
구체적인 일 태양에서, 상기 양친성 블록 공중합체는, 친수성 A 블록 및 소수성 B 블록을 포함하는 A-B 형 블록 공중합체일 수 있다. 상기 A-B 형 블록 공중합체는, 수용액 상에서, 소수성 B 블록이 코어(내벽)를 형성하고 친수성 A 블록이 쉘(외벽)을 형성하는 코어-쉘 타입의 고분자 나노입자를 형성한다. In a specific embodiment, the amphiphilic block copolymer may be an A-B type block copolymer including a hydrophilic A block and a hydrophobic B block. The A-B type block copolymer forms core-shell type polymer nanoparticles in which the hydrophobic B block forms a core (inner wall) and the hydrophilic A block forms a shell (outer wall) in an aqueous solution.
일 구체예에서, 상기 친수성 A 블록은 폴리알킬렌글리콜, 폴리비닐알콜, 폴리비닐피롤리돈, 폴리아크릴아미드 및 그 유도체로 구성된 군으로부터 선택되는 하나 이상일 수 있다. In one embodiment, the hydrophilic A block may be at least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, and derivatives thereof.
보다 구체적으로, 상기 친수성 A 블록은 모노메톡시폴리에틸렌클리콜, 모노아세톡시폴리에틸렌글리콜, 폴리에틸렌글리콜, 폴리에틸렌과 프로필렌글리콜의 공중합체 및 폴리비닐피롤리돈으로 구성된 군으로부터 선택되는 하나 이상일 수 있다. More specifically, the hydrophilic block A may be at least one selected from the group consisting of monomethoxypolyethylene glycol, monoacetoxypolyethylene glycol, polyethylene glycol, a copolymer of polyethylene and propylene glycol, and polyvinylpyrrolidone.
일 구체예에서, 상기 친수성 A 블록은 수평균분자량이 200 내지 50,000달톤인 것일 수 있고, 보다 구체적으로는 1,000 내지 20,000달톤, 보다 더 구체적으로는 1,000 내지 5,000달톤인 것일 수 있다. In one embodiment, the hydrophilic A block may have a number average molecular weight of 200 to 50,000 Daltons, more specifically 1,000 to 20,000 Daltons, and even more specifically 1,000 to 5,000 Daltons.
또한, 필요에 따라, 상기 친수성 A 블록의 말단에 특정 조직이나 세포에 도달할 수 있는 작용기, 리간드, 또는 세포내 전달을 촉진할 수 있는 작용기를 화학적으로 결합시켜 양친성 블록 공중합체와 폴리락트산염으로 형성된 고분자 나노입자 전달체의 체내 분포를 조절하거나 상기 나노입자 전달체가 세포 내로 전달되는 효율을 높일 수 있다. 일 구체예에서, 상기 작용기나 리간드는 단당류, 다당류, 비타민, 펩타이드, 단백질 및 세포 표면 수용체에 대한 항체로 이루어진 군에서 선택된 1종 이상일 수 있다. 보다 구체적으로, 상기 작용기나 리간드는 아니사마이드(anisamide), 비타민 B9(엽산), 비타민 B12, 비타민A, 갈락토오스, 락토오스, 만노오스, 히알루론산, RGD 펩타이드, NGR 펩타이드, 트랜스페린, 트랜스페린 수용체에 대한 항체 등으로 이루어진 군에서 선택된 1종 이상일 수 있다.In addition, if necessary, the amphiphilic block copolymer and polylactate are chemically bound to the end of the hydrophilic A block with a functional group capable of reaching a specific tissue or cell, a ligand, or a functional group capable of facilitating intracellular delivery. It is possible to control the distribution in the body of the polymer nanoparticle delivery system formed with the above, or to increase the efficiency of the delivery of the nanoparticle delivery system into cells. In one embodiment, the functional group or ligand may be at least one selected from the group consisting of monosaccharides, polysaccharides, vitamins, peptides, proteins, and antibodies to cell surface receptors. More specifically, the functional group or ligand is anisamide, vitamin B9 (folic acid), vitamin B12, vitamin A, galactose, lactose, mannose, hyaluronic acid, RGD peptide, NGR peptide, transferrin, antibody against transferrin receptor It may be one or more selected from the group consisting of.
상기 소수성 B 블록은 생체적합성 생분해성 고분자로서, 일 구체예에서, 이는 폴리에스테르, 폴리언하이드라이드, 폴리아미노산, 폴리오르소에스테르 및 폴리포스파진으로 구성된 군으로부터 선택되는 하나 이상일 수 있다. The hydrophobic B block is a biocompatible biodegradable polymer, and in one embodiment, it may be one or more selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester, and polyphosphazine.
보다 구체적으로, 상기 소수성 B 블록은 폴리락타이드, 폴리글리콜라이드, 폴리카프로락톤, 폴리디옥산-2-온, 폴리락타이드와 글리콜라이드의 공중합체, 폴리락타이드와 폴리디옥산-2-온의 공중합체, 폴리락타이드와 폴리카프로락톤의 공중합체 및 폴리글리콜라이드와 폴리카프로락톤의 공중합체로 구성된 군으로부터 선택되는 하나 이상일 수 있다. More specifically, the hydrophobic B block is polylactide, polyglycolide, polycaprolactone, polydioxan-2-one, a copolymer of polylactide and glycolide, polylactide and polydioxan-2-one It may be at least one selected from the group consisting of a copolymer of, a copolymer of polylactide and polycaprolactone, and a copolymer of polyglycolide and polycaprolactone.
일 구체예에서, 상기 소수성 B 블록은 수평균분자량이 50 내지 50,000달톤인 것일 수 있고, 보다 구체적으로는 200 내지 20,000달톤, 보다 더 구체적으로는 1,000 내지 5,000달톤인 것일 수 있다. In one embodiment, the hydrophobic B block may have a number average molecular weight of 50 to 50,000 Daltons, more specifically 200 to 20,000 Daltons, and even more specifically 1,000 to 5,000 Daltons.
또한, 일 구체예에서, 상기 소수성 B 블록은, 소수성 B 블록의 소수성을 증가시켜 나노입자의 안정성을 향상시키기 위하여, 소수성 B 블록 말단의 히드록시기에 토코페롤, 콜레스테롤, 또는 탄소수 10 내지 24개의 지방산을 화학적으로 결합시키는 것에 의하여 수식된 것일 수 있다. In addition, in one embodiment, the hydrophobic B block, in order to increase the hydrophobicity of the hydrophobic B block to improve the stability of the nanoparticles, tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms in the hydroxy group at the end of the hydrophobic B block is chemically It may be modified by combining with .
일 구체예에서, 상기 양친성 블록 공중합체에 있어서, 친수성 블록(A)과 소수성 블록(B)의 조성비는, 공중합체 총중량을 기준으로, 친수성 블록(A)이 40 내지 70중량% 범위일 수 있고, 보다 구체적으로는 50 내지 60중량% 범위일 수 있다. 친수성 블록(A)의 비율이 공중합체 총중량을 기준으로 40중량% 미만이면 고분자의 물에 대한 용해도가 낮아서 나노입자를 형성하기 어렵기 때문에, 공중합체가 나노입자를 형성하기에 충분한 물에 대한 용해도를 갖기 위하여 친수성 블록(A)의 비율이 40중량% 이상인 것이 좋다. 한편, 친수성 블록(A)의 비율이 공중합체 총중량을 기준으로 70중량%를 초과하면 친수성이 너무 높아 고분자 나노입자의 안정성이 낮아져서 유효성분/양이온성 화합물 복합체의 가용화 조성물로 사용하기 어려우므로, 나노입자 안정성을 고려하여 친수성 블록(A)의 비율이 70중량% 이하인 것이 좋다.In one embodiment, in the amphiphilic block copolymer, the composition ratio of the hydrophilic block (A) and the hydrophobic block (B), based on the total weight of the copolymer, the hydrophilic block (A) may be in the range of 40 to 70% by weight. And, more specifically, it may be in the range of 50 to 60% by weight. If the proportion of the hydrophilic block (A) is less than 40% by weight based on the total weight of the copolymer, the solubility of the polymer in water is low and it is difficult to form nanoparticles, so that the copolymer has sufficient solubility in water to form nanoparticles. It is preferable that the proportion of the hydrophilic block (A) is 40% by weight or more in order to have a On the other hand, if the proportion of the hydrophilic block (A) exceeds 70% by weight based on the total weight of the copolymer, the hydrophilicity is too high and the stability of the polymer nanoparticles is lowered, so it is difficult to use it as a solubilization composition of the active ingredient/cationic compound complex, In consideration of particle stability, it is preferable that the proportion of the hydrophilic block (A) is 70% by weight or less.
일 구체예에서, 본 발명의 키트에 의하여 형성되는 조성물 내의 상기 폴리락트산염은 나노입자의 코어(내벽)에 분포하여 코어의 소수성을 강화시켜 나노입자를 안정시킴과 동시에 체내에서 세망내피계(RES)를 효과적으로 회피하는 역할을 한다. 즉, 폴리락트산염의 카르복실산 음이온이 폴리락트산보다 효과적으로 양이온성 복합체와 결합하여 고분자 나노입자의 표면전위를 감소시켜 폴리락트산염을 포함하지 않는 고분자 나노입자에 비해 표면전위의 양성 전하가 감소하여 세망내피계에 의해 덜 포획되고, 이로 인하여 목적하는 부위(예컨대, 암세포, 염증세포 등)로의 전달 효율이 우수하다는 장점이 있다.In one embodiment, the polylactic acid salt in the composition formed by the kit of the present invention is distributed in the core (inner wall) of the nanoparticles to strengthen the hydrophobicity of the core to stabilize the nanoparticles and at the same time, the reticuloendothelial system (RES) in the body ) to effectively avoid That is, the carboxylate anion of polylactic acid binds to the cationic complex more effectively than polylactic acid and reduces the surface potential of the polymer nanoparticles. It is less captured by the endothelial system, and thus has the advantage of excellent delivery efficiency to a target site (eg, cancer cells, inflammatory cells, etc.).
일 구체예에서, 상기 양친성 블록 공중합체와는 별도의 성분으로서 나노입자 내벽 성분으로 포함되는 폴리락트산염은 수평균분자량이 500 내지 50,000달톤인 것일 수 있고, 보다 구체적으로는 1,000 내지 10,000달톤인 것일 수 있다. 폴리락트산염의 수평균분자량이 500달톤 미만이면 소수성이 너무 낮아 나노입자의 코어(내벽)에 존재하기 어려울 수 있고, 50,000달톤을 초과하면 고분자 나노입자의 입자가 커지는 문제가 있을 수 있다.In one embodiment, as a separate component from the amphiphilic block copolymer, the polylactic acid salt included as a component of the inner wall of the nanoparticles may have a number average molecular weight of 500 to 50,000 Daltons, and more specifically, 1,000 to 10,000 Daltons. it could be If the number average molecular weight of the polylactate is less than 500 Daltons, hydrophobicity may be too low to exist in the core (inner wall) of the nanoparticles, and if it exceeds 50,000 Daltons, there may be a problem in that the particles of the polymer nanoparticles become large.
일 구체예에서, 상기 폴리락트산염(예컨대, 폴리락트산나트륨염)의 말단 중 카르복실산금속(예컨대, 카르복실산나트륨)의 반대편의 말단은, 히드록시, 아세톡시, 벤조일옥시, 데카노일옥시, 팔미토일옥시 및 탄소수 1 내지 2개의 알콕시로 이루어진 그룹 중에서 선택된 하나로 치환될 수 있다.In one embodiment, the terminal opposite to the metal carboxylate (eg, sodium carboxylate) among the ends of the polylactate (eg, sodium polylactate) is hydroxy, acetoxy, benzoyloxy, decanoyloxy , may be substituted with one selected from the group consisting of palmitoyloxy and alkoxy having 1 to 2 carbon atoms.
일 구체예에서, 상기 폴리락트산염은 하기 화학식 2 내지 7의 화합물로 이루어진 군으로부터 선택되는 하나 이상일 수 있다(여기서 “COO”는 카르복실기, 즉 “C(=O)O”를 의미한다):In one embodiment, the polylactic acid salt may be one or more selected from the group consisting of compounds of Formulas 2 to 7 (herein, “COO” means a carboxyl group, that is, “C(=O)O”):
[화학식 2][Formula 2]
RO-CHZ-[A]m-[B]n-COOMRO-CHZ-[A] m -[B] n -COOM
상기 식 2에서, A는 -COO-CHZ-이고; B는 -COO-CHY-, -COO-CH2CH2CH2CH2CH2- 또는 -COO-CH2CH2OCH2-이며; R은 수소원자, 또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸, 또는 에틸기이고; Z와 Y는 각각 수소원자, 또는 메틸 또는 페닐기이며; M은 Na, K, 또는 Li이고; n은 1 내지 30의 정수이며; m은 0 내지 20의 정수이다;In Formula 2, A is -COO-CHZ-; B is -COO-CHY-, -COO-CH 2 CH 2 CH 2 CH 2 CH 2 - or -COO-CH 2 CH 2 OCH 2 -; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl, or ethyl group; Z and Y are each a hydrogen atom, or a methyl or phenyl group; M is Na, K, or Li; n is an integer from 1 to 30; m is an integer from 0 to 20;
[화학식 3][Formula 3]
RO-CHZ-[COO-CHX]p-[COO-CHY']q-COO-CHZ-COOMRO-CHZ-[COO-CHX] p -[COO-CHY'] q -COO-CHZ-COOM
상기 식 3에서, X는 메틸기이고; Y'는 수소원자 또는 페닐기이며; p는 0 내지 25의 정수이고, q는 0 내지 25의 정수이되, 단 p+q는 5 내지 25의 정수이고; R은 수소원자, 또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸 또는 에틸기이며; M은 Na, K, 또는 Li이고; Z 는 수소 원자, 메틸 또는 페닐기이다;In Formula 3, X is a methyl group; Y' is a hydrogen atom or a phenyl group; p is an integer from 0 to 25, q is an integer from 0 to 25, with the proviso that p+q is an integer from 5 to 25; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is Na, K, or Li; Z is a hydrogen atom, a methyl or phenyl group;
[화학식 4][Formula 4]
RO-PAD-COO-W-M'RO-PAD-COO-W-M'
상기 식 4에서, W-M'는
Figure PCTKR2020018480-appb-I000002
또는
Figure PCTKR2020018480-appb-I000003
이고; PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공중합체, D,L-락트산과 만델릭산의 공중합체, D,L-락트산과 카프로락톤의 공중합체 및 D,L-락트산과 1,4-디옥산-2-온의 공중합체로 구성된 그룹으로부터 선택되는 것이며; R은 수소원자, 또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸 또는 에틸기이고; M은 독립적으로 Na, K, 또는 Li이다;In Equation 4, W-M' is
Figure PCTKR2020018480-appb-I000002
or
Figure PCTKR2020018480-appb-I000003
ego; PAD is D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic acid, a copolymer of D,L-lactic acid and mandelic acid, D,L-lactic acid and copolymers of caprolactone and copolymers of D,L-lactic acid and 1,4-dioxan-2-one; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is independently Na, K, or Li;
[화학식 5][Formula 5]
S-O-PAD-COO-QS-O-PAD-COO-Q
상기 식 5에서, S는
Figure PCTKR2020018480-appb-I000004
이고; L은 -NR1- 또는 -0-이며, 여기서 R1은 수소원자 또는 C1-10알킬이고; Q는 CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2CH3, 또는 CH2C6H5이고; a는 0 내지 4의 정수이며; b는 1 내지 10의 정수이고; M은 Na, K, 또는 Li이며; PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공중합체, D,L-락트산과 만델릭산의 공중합체, D,L-락트산과 카프로락톤의 공중합체, 및 D,L-락트산과 1,4-디옥산-2-온의 공중합체로 이루어진 군에서 선택되는 하나 이상이다;In Equation 5, S is
Figure PCTKR2020018480-appb-I000004
ego; L is -NR 1 - or -0-, wherein R 1 is a hydrogen atom or C 1-10 alkyl; Q is CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH 2 CH 2 CH 2 CH 3 , or CH 2 C 6 H 5 ; a is an integer from 0 to 4; b is an integer from 1 to 10; M is Na, K, or Li; PAD is D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic acid, a copolymer of D,L-lactic acid and mandelic acid, D,L-lactic acid and at least one selected from the group consisting of a copolymer of caprolactone, and a copolymer of D,L-lactic acid and 1,4-dioxan-2-one;
[화학식 6][Formula 6]
Figure PCTKR2020018480-appb-I000005
또는
Figure PCTKR2020018480-appb-I000006
Figure PCTKR2020018480-appb-I000005
or
Figure PCTKR2020018480-appb-I000006
상기 식 6에서, R'는 -PAD-O-C(O)-CH2CH2-C(O)-OM이고, 여기서 PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공중합체, D,L-락트산과 만델릭산의 공중합체, D,L-락트산과 카프로락톤의 공중합체, D,L-락트산과 1,4-디옥산-2-온의 공중합체로 구성된 그룹으로부터 선택되는 것이고, M은 Na, K, 또는 Li이며; a는 1 내지 4의 정수이다;In Formula 6, R' is -PAD-OC(O)-CH 2 CH 2 -C(O)-OM, wherein PAD is D,L-polylactic acid, D-polylactic acid, polymandelic acid, D ,L-lactic acid and glycolic acid copolymer, D,L-lactic acid and mandelic acid copolymer, D,L-lactic acid and caprolactone copolymer, D,L-lactic acid and 1,4-dioxane-2 -one is selected from the group consisting of copolymers, M is Na, K, or Li; a is an integer from 1 to 4;
[화학식 7][Formula 7]
YO-[-C(O)-(CHX)a-O-]m-C(O)-R-C(O)-[-O-(CHX')b-C(O)-]n-OZYO-[-C(O)-(CHX) a -O-] m -C(O)-RC(O)-[-O-(CHX') b -C(O)-] n -OZ
상기 식 7에서, X 및 X'은 독립적으로 수소, 탄소수가 1~10인 알킬 또는 탄소수가 6~20인 아릴이고; Y 및 Z는 독립적으로 Na, K, 또는 Li이며; m 및 n은 독립적으로 0 내지 95의 정수이되, 5 < m + n < 100이고; a 및 b는 독립적으로 1 내지 6의 정수이며; R은 -(CH2)k-, 탄소수가 2~10인 2가 알케닐(divalent alkenyl), 탄소수가 6~20인 2가 아릴(divalent aryl) 또는 이들의 조합이고, 여기서 k는 0 내지 10의 정수이다.In Formula 7, X and X' are independently hydrogen, alkyl having 1 to 10 carbons, or aryl having 6 to 20 carbons; Y and Z are independently Na, K, or Li; m and n are independently integers from 0 to 95, provided that 5 < m + n <100; a and b are independently integers from 1 to 6; R is -(CH 2 ) k -, divalent alkenyl having 2 to 10 carbon atoms, divalent aryl having 6 to 20 carbon atoms, or a combination thereof, where k is 0 to 10 is the integer of
일 구체예에서, 상기 폴리락트산염은 상기 화학식 2 또는 화학식 3의 화합물일 수 있다.In one embodiment, the polylactate may be a compound of Formula 2 or Formula 3 above.
일 구체예에서, 본 발명의 키트는, mRNA의 세포 내 전달 효율을 증가시키기 위하여, 본 발명의 키트에 의하여 형성되는 조성물의 총중량을 기준으로 0.01 내지 50중량%의, 보다 구체적으로는 0.1 내지 10중량%의 융합성 지질을 추가로 포함할 수 있다.In one embodiment, the kit of the present invention, in order to increase the intracellular delivery efficiency of mRNA, 0.01 to 50% by weight based on the total weight of the composition formed by the kit of the present invention, more specifically 0.1 to 10 It may further comprise fusible lipids in weight percent.
상기 융합성 지질은 mRNA와 양이온성 지질의 복합체에 혼합시, 소수성 상호작용으로 결합하여 mRNA, 양이온성 지질 및 융합성 지질의 복합체를 형성하고, 상기 융합성 지질을 포함하는 복합체는 양친성 블록 공중합체의 나노입자 구조 내부에 봉입된다. When the fusible lipid is mixed with the complex of mRNA and cationic lipid, it binds by hydrophobic interaction to form a complex of mRNA, cationic lipid and fused lipid, and the complex including the fused lipid is amphiphilic block air Encapsulated within the composite nanoparticle structure.
일 구체예에서, 상기 융합성 지질은 인지질, 콜레스테롤, 및 토코페롤로 구성된 군으로부터 선택된 하나 또는 둘 이상의 조합일 수 있다. In one embodiment, the fusible lipid may be one or a combination of two or more selected from the group consisting of phospholipids, cholesterol, and tocopherol.
보다 구체적으로, 상기 인지질은 포스파티딜에탄올아민(phosphatidylethanolamin, PE), 포스파티딜콜린(phosphatidylcholine, PC) 및 포스파티딘산(phosphatidic acid)으로 이루어진 군에서 선택된 1종 이상일 수 있다. 상기 포스파티딜에탄올아민(phosphatidylethanolamin, PE), 포스파티딜콜린(phosphatidylcholine, PC) 및 포스파티딘산은 하나 또는 2 개의 C10-24 지방산과 결합된 형태일 수 있다. 상기 콜레스테롤 및 토코페롤에는 콜레스테롤 및 토코페롤의 각 유사체, 유도체, 및 대사체가 포함된다. More specifically, the phospholipid may be at least one selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidic acid. The phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidic acid may be combined with one or two C10-24 fatty acids. The cholesterol and tocopherols include each analog, derivative, and metabolite of cholesterol and tocopherol.
보다 더 구체적으로, 상기 융합성 지질은 디라우로일 포스파티딜에탄올아민(dilauroyl phosphatidylethanolamine), 디미리스토일 포스파티딜에탄올아민(dimyristoyl phosphatidylethanolamine), 디팔미토일 포스파티딜에탄올아민(dipalmitoyl phosphatidylethanolamine), 디스테아로일 포스파티딜에탄올아민(distearoyl phosphatidylethanolamine), 디올레오일 포스파티딜에탄올아민(dioleoyl phosphatidylethanolamine, DOPE), 디팔미토올레오일 포스포에탄올아민(1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, DPPE), 디리놀레오일 포스파티딜에탄올아민(dilinoleoyl phosphatidylethanolamine), 1-팔미토일-2-올레오일 포스파티딜에탄올아민(1-palmitoyl-2-oleoyl phosphatidylethanolamine), 1,2-디피타노일-3-sn-포스파티딜에탄올아민(1,2-diphytanoyl-3-sn-phosphatidylethanolamine), 디팔미토올레오일 포스포콜린(1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, DPPC), 디올레오일 포스포콜린(1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 디라우로일 포스파티딜콜린(dilauroyl phosphatidylcholine), 디미리스토일 포스파티딜콜린(dimyristoyl phosphatidylcholine), 디팔미토일 포스파티딜콜린(dipalmitoyl phosphatidylcholine), 디스테아로일 포스파티딜콜린(distearoyl phosphatidylcholine), 디올레오일 포스파티딜콜린(dioleoyl phosphatidylcholine), 디리놀레오일 포스파티딜콜린(dilinoleoyl phosphatidylcholine), 1-팔미토일-2-올레오일 포스파티딜콜린(1-palmitoyl-2-oleoyl phosphatidylcholine), 1,2-디피타노일-3-sn-포스파티딜콜린(1,2-diphytanoyl-3-sn-phosphatidylcholine), 디라우로일 포스파티딘산(dilauroyl phosphatidic acid), 디미리스토일 포스파티딘산(dimyristoyl phosphatidic acid), 디팔미토일 포스파티딘산(dipalmitoyl phosphatidic acid), 디스테아로일 포스파티딘산(distearoyl phosphatidic acid), 디올레오일 포스파티딘산(dioleoyl phosphatidic acid), 디리놀레오일 포스파티딘산(dilinoleoyl phosphatidic acid), 1-팔미토일-2-올레오일 포스파티딘산(1-palmitoyl-2-oleoyl phosphatidic acid), 1,2-디피타노일-3-sn-포스파티딘산(1,2-diphytanoyl-3-sn-phosphatidic acid), 콜레스테롤 및 토코페롤로 구성된 군으로부터 선택된 하나 또는 둘 이상의 조합일 수 있다. More specifically, the fusible lipids include dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, and distearoyl Ethanolamine (distearoyl phosphatidylethanolamine), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoleoyl phosphoethanolamine (1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, DPPE), dilinoleo yl phosphatidylethanolamine (dilinoleoyl phosphatidylethanolamine), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (1-palmitoyl-2-oleoyl phosphatidylethanolamine), 1,2-dipitanoyl-3-sn-phosphatidylethanolamine (1, 2-diphytanoyl-3-sn-phosphatidylethanolamine), dipalmitoleoyl phosphocholine (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, DPPC), dioleoyl phosphocholine (1,2-dioleoyl- sn-glycero-3-phosphocholine, DOPC), dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dispalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine dioleoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine ), 1-palmitoyl-2-oleoyl phosphatidylcholine (1-palmitoyl-2-oleoyl phosphatidylcholine), 1,2-diphytanoyl-3-sn-phosphatidylcholine (1,2-diphytanoyl-3-sn-phosphatidylcholine), Dilauroyl phosphatidic acid, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid , dioleoyl phosphatidic acid, dilinoleoyl phosphatidic acid, 1-palmitoyl-2-oleoyl phosphatidic acid, It may be one or a combination of two or more selected from the group consisting of 1,2-diphytanoyl-3-sn-phosphatidic acid, cholesterol and tocopherol.
보다 더 구체적으로, 상기 융합성 지질은 디올레오일 포스파티딜에탄올아민(dioleoyl phosphatidylethanolamine, DOPE), 디팔미토올레오일 포스포콜린(1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, DPPC), 디올레오일 포스포콜린(1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) 및 디팔미토올레오일 포스포에탄올아민(1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, DPPE)으로 이루어진 군에서 선택된 1종 이상일 수 있다.More specifically, the fusible lipid is dioleoyl phosphatidylethanolamine (DOPE), dipalmitoleoyl phosphocholine (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, DPPC), diol as leoyl phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) and dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE) It may be one or more selected from the group consisting of.
일 구체예에서, 본 발명의 키트에 의하여 형성되는 조성물 내의 구성 성분 중, 상기 친수성 블록(A)과 소수성 블록(B)을 포함하는 양친성 블록 공중합체의 함량은, 상기 양이온성 화합물 1 중량부를 기준으로, 0.01 내지 50 중량부, 0.01 내지 30 중량부, 0.01 내지 15 중량부, 0.01 내지 12 중량부, 0.1 내지 50 중량부, 0.1 내지 30 중량부, 0.1 내지 15 중량부, 0.1 내지 12 중량부, 0.5 내지 50 중량부, 0.5 내지 30 중량부, 0.5 내지 15 중량부, 0.5 내지 12 중량부, 1 내지 50 중량부, 1 내지 30 중량부, 1 내지 15 중량부, 또는 1 내지 12 중량부일 수 있다. In one embodiment, among the components in the composition formed by the kit of the present invention, the content of the amphiphilic block copolymer including the hydrophilic block (A) and the hydrophobic block (B) is 1 part by weight of the cationic compound based on 0.01 to 50 parts by weight, 0.01 to 30 parts by weight, 0.01 to 15 parts by weight, 0.01 to 12 parts by weight, 0.1 to 50 parts by weight, 0.1 to 30 parts by weight, 0.1 to 15 parts by weight, 0.1 to 12 parts by weight , 0.5 to 50 parts by weight, 0.5 to 30 parts by weight, 0.5 to 15 parts by weight, 0.5 to 12 parts by weight, 1 to 50 parts by weight, 1 to 30 parts by weight, 1 to 15 parts by weight, or 1 to 12 parts by weight. have.
보다 구체적으로, 상기 양친성 블록 공중합체의 함량은 상기한 범위 내에서 유효성분에 따라 조절될 수 있다. 예를 들어, 일 구체예에서 유효성분이 바이러스인 경우, 상기 양친성 블록 공중합체의 함량은, 양이온성 화합물 1 중량부를 기준으로 3 내지 7 중량부일 수 있고, 다른 구체예에서 유효성분이 핵산인 경우, 상기 양친성 블록 공중합체의 함량은, 양이온성 화합물 1 중량부를 기준으로 0.7 내지 7 중량부, 1 내지 5 중량부, 또는 1 내지 3 중량부일 수 있다.More specifically, the content of the amphiphilic block copolymer may be adjusted according to the active ingredient within the above range. For example, when the active ingredient is a virus in one embodiment, the content of the amphiphilic block copolymer may be 3 to 7 parts by weight based on 1 part by weight of the cationic compound, and in another embodiment, when the active ingredient is a nucleic acid, The content of the amphiphilic block copolymer may be 0.7 to 7 parts by weight, 1 to 5 parts by weight, or 1 to 3 parts by weight based on 1 part by weight of the cationic compound.
일 구체예에서, 본 발명의 키트에 의하여 형성되는 조성물 내의 구성 성분 중, 상기 폴리락트산염의 함량은, 상기 양이온성 화합물 1 중량부를 기준으로, 0.1 내지 100 중량부, 0.1 내지 80 중량부, 0.1 내지 50 중량부, 0.1 내지 30 중량부, 0.1 내지 10 중량부, 0.1 내지 5 중량부, 0.5 내지 100 중량부, 0.5 내지 80 중량부, 0.5 내지 50 중량부, 0.5 내지 30 중량부, 1 내지 100 중량부, 1 내지 80 중량부, 1 내지 50 중량부, 1 내지 30 중량부, 2 내지 100 중량부, 2 내지 80 중량부, 2 내지 50 중량부, 또는 2 내지 30 중량부일 수 있다. In one embodiment, among the components in the composition formed by the kit of the present invention, the content of the polylactic acid salt is 0.1 to 100 parts by weight, 0.1 to 80 parts by weight, 0.1 to 100 parts by weight, based on 1 part by weight of the cationic compound. 50 parts by weight, 0.1 to 30 parts by weight, 0.1 to 10 parts by weight, 0.1 to 5 parts by weight, 0.5 to 100 parts by weight, 0.5 to 80 parts by weight, 0.5 to 50 parts by weight, 0.5 to 30 parts by weight, 1 to 100 parts by weight parts, 1 to 80 parts by weight, 1 to 50 parts by weight, 1 to 30 parts by weight, 2 to 100 parts by weight, 2 to 80 parts by weight, 2 to 50 parts by weight, or 2 to 30 parts by weight.
보다 구체적으로, 상기 폴리락트산염의 함량은 상기한 범위 내에서 유효성분에 따라 조절될 수 있다. 예를 들어, 일 구체예에서 유효성분이 바이러스인 경우, 상기 폴리락트산염의 함량은, 양이온성 화합물 1 중량부를 기준으로 3 내지 15 중량부일 수 있고, 다른 구체예에서 유효성분이 핵산인 경우, 상기 폴리락트산염의 함량은, 양이온성 화합물 1 중량부를 기준으로 0.2 내지 4 중량부, 1 내지 4 중량부 또는 0.2 내지 0.8 중량부일 수 있다.More specifically, the content of the polylactic acid salt may be adjusted according to the active ingredient within the above range. For example, in one embodiment, when the active ingredient is a virus, the content of the polylactic acid salt may be 3 to 15 parts by weight based on 1 part by weight of the cationic compound, and in another embodiment, when the active ingredient is a nucleic acid, the polylactic acid The content of the salt may be 0.2 to 4 parts by weight, 1 to 4 parts by weight, or 0.2 to 0.8 parts by weight based on 1 part by weight of the cationic compound.
일 구체예에서, 상기 제1 챔버 및/또는 제2 챔버는 수성 용액, 수혼화성 유기용매 또는 이들의 조합을 추가로 포함할 수 있다. 상기 “수성 용액”은 수용액과 같은 의미로 사용될 수 있고, 예를 들어, 물, 멸균정제수, 완충액, 주사액 등을 의미할 수 있으며, 유기산을 더 포함한 완충액일 수도 있다. 상기 수성 용액은 예를 들어, 시트르산 완충액, PBS 완충액 등일 수 있으나, 이에 제한되지 않는다. 상기 “수혼화성 유기용매”는 C1 내지 C4의 저급 알코올, 아세톤, 아세토니트릴, 이의 수혼합물 또는 이들의 혼합물일 수 있으나, 이에 제한되지 않는다.In one embodiment, the first chamber and/or the second chamber may further include an aqueous solution, a water-miscible organic solvent, or a combination thereof. The "aqueous solution" may be used in the same sense as an aqueous solution, for example, may refer to water, sterile purified water, buffer solution, injection solution, etc., may be a buffer solution further containing an organic acid. The aqueous solution may be, for example, a citric acid buffer, a PBS buffer, and the like, but is not limited thereto. The "water-miscible organic solvent" may be a C1 to C4 lower alcohol, acetone, acetonitrile, a water mixture thereof, or a mixture thereof, but is not limited thereto.
일 구체예에서, 상기 제1 챔버에 포함된 양친성 블록 공중합체, 양이온성 화합물, 및 폴리락트산염의 혼합물은 혼합 후 친수성 필터로 1회 이상 여과하여 제1 챔버의 내용물로서 이용되는 것일 수 있다. 친수성 필터의 재질로는 예를 들어, 나일론, 혼합형 셀룰로오스 에스테르(Mixed Cellulose Ester, MCE), 폴리에틸설폰(PES), 폴리비닐리덴 디플루오라이드(polyvinylidene difluoridem PVDF), 셀룰로오스 아세테이트(cellulose acetate, CA), 폴리테트라플루오로에틸렌(PTFE), 및 이들의 혼합물이 있으나 이에 제한되지 않는다. 친수성 필터한 경우, 유효성분이 보다 성공적으로 나노입자 내에 포합될 수 있고, 나노입자의 안정성이 증가할 수 있다.In one embodiment, the mixture of the amphiphilic block copolymer, the cationic compound, and the polylactic acid salt contained in the first chamber may be used as the contents of the first chamber by filtering one or more times with a hydrophilic filter after mixing. The material of the hydrophilic filter is, for example, nylon, mixed cellulose ester (MCE), polyethylsulfone (PES), polyvinylidene difluoridem PVDF, cellulose acetate (CA). , polytetrafluoroethylene (PTFE), and mixtures thereof. In the case of a hydrophilic filter, the active ingredient may be more successfully incorporated into the nanoparticles, and the stability of the nanoparticles may be increased.
일 구체예에서, 상기 제2 챔버는 유효성분의 안정성을 향상시키는데 적절한 안정화제를 추가로 포함할 수 있다. 상기 안정화제는, pH 조절제, 무기염, 당류, 계면활성제, 킬레이트제 등을 더 포함할 수 있으나 이에 제한되지 않는다. 상기 “당류”는 단당류, 이당류, 이들의 환원당인 당알코올, 단일 또는 혼합된 다수 당의 폴리머 등을 의미할 수 있고, 상기 다당류는 3당류 이상 당류를 의미할 수 있다. 상기 단당류는, 예를 들어, 만노스, 글루코스, 아라비노스, 프럭토스, 갈락토스 등이 있고; 상기 이당류로는, 수크로스, 트레할로스, 말토스, 락토스, 셀로비오스, 겐티오비오스, 이소말토스, 멜리보우스 등이 있고; 상기 당알코올은 만니톨, 소르비톨, 자일리톨, 에리스리톨, 말티톨 등이 있고; 상기 다당류로는, 라피노스, 덱스트란, 전분, 히드록시에틸 전분, 시클로덱스트린, 셀룰로스, 헤타스타치, 올리고당이 있으나, 이에 제한되지 않는다. 상기 “pH-조절제”는 트리스(Tris), 글라이신, 히스티딘, 글루타메이트, 숙시네이트, 포스페이트, 아세테이트, 아스파르테이트 또는 이들의 조합일 수 있고, 상기 “계면활성제”는 나트륨 라우릴 술페이트, 디옥틸 나트륨 술포숙시네이트, 디옥틸나트륨 술포네이트, 케노데옥시콜산, N-라우로일사르코신 나트륨염, 리튬 도데실 술페이트, 1-옥탄술폰산 나트륨염, 나트륨 콜레이트 수화물, 나트륨 데옥시콜레이트, 글리코데옥시콜산 나트륨염, 벤즈알코늄 클로라이드, 트리톤(Triton) X-100, 트리톤 X-114, 라우로마크로골(lauromacrogol) 400, 폴리옥실 40 스테아레이트, 폴리소르베이트 20, 40, 60, 65 및 80 또는 이들의 조합일 수 있으나, 이에 제한되지 않는다. 상기 “킬레이트제”는 구연산, 폴리페놀릭산, EDTA, DTPA, EDDHA 또는 이들의 조합일 수 있으나, 이에 제한되지 않는다. 상기 “무기염”은 1가 또는 2가 금속의 염을 의미하는 것으로서, NaCl, KCl, MgCl2, CaCl2, MgSO4, CaSO4, CaCO3, MgCO3 등일 수 있으나 이에 제한되지 않는다.In one embodiment, the second chamber may further include a stabilizer suitable for improving the stability of the active ingredient. The stabilizer may further include a pH adjuster, an inorganic salt, a saccharide, a surfactant, a chelating agent, and the like, but is not limited thereto. The “saccharide” may refer to monosaccharides, disaccharides, sugar alcohols that are reducing sugars thereof, polymers of single or mixed polysaccharides, and the like, and the polysaccharides may refer to three or more saccharides. The monosaccharides include, for example, mannose, glucose, arabinose, fructose, galactose, and the like; Examples of the disaccharide include sucrose, trehalose, maltose, lactose, cellobiose, gentiobiose, isomaltose, melibose, and the like; The sugar alcohol includes mannitol, sorbitol, xylitol, erythritol, maltitol, and the like; Examples of the polysaccharide include, but are not limited to, raffinose, dextran, starch, hydroxyethyl starch, cyclodextrin, cellulose, hetastarch, and oligosaccharide. The “pH-adjusting agent” may be Tris, glycine, histidine, glutamate, succinate, phosphate, acetate, aspartate or a combination thereof, and the “surfactant” may be sodium lauryl sulfate, dioctyl Sodium sulfosuccinate, dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, glycoside Deoxycholic acid sodium salt, benzalkonium chloride, Triton X-100, Triton X-114, lauromacrogol 400, polyoxyl 40 stearate, polysorbate 20, 40, 60, 65 and 80 or a combination thereof, but is not limited thereto. The “chelating agent” may be citric acid, polyphenolic acid, EDTA, DTPA, EDDHA, or a combination thereof, but is not limited thereto. The “inorganic salt” refers to a salt of a monovalent or divalent metal, and may be NaCl, KCl, MgCl 2 , CaCl 2 , MgSO 4 , CaSO 4 , CaCO 3 , MgCO 3 , or the like, but is not limited thereto.
예를 들어, 유효성분이 바이러스인 경우, 상기 제2 챔버는 5 내지 15 mM Tris, 5 내지 15 mM 히스티딘, 50 내지 90 mM NaCl, 2 내지 8% 수크로오스 (w/v), 0.5 내지 1.5 mM MgCl2, 0.005 내지 0.05% (w/v) PS-80, 0.05 내지 0.15 mM EDTA, 및 0.1 내지 1.0% 에탄올 (v/v)를 더 포함할 수 있고, pH는 7.0 내지 8.0일 수 있다. 다른 구체예에서, 유효성분이 핵산인 경우, 상기 제2 챔버는 PBS 완충액, 예를 들어, PH 7.0 내지 pH 8.0, 2.0 내지 3.5 mM KCl, 1.0 내지 2.5mM KH2PO4, 125 내지 145 mM NaCl, 및 7.5 내지 9.5 mM Na2HPO4를 포함하는 용액을 더 포함할 수 있다.For example, when the active ingredient is a virus, the second chamber is 5 to 15 mM Tris, 5 to 15 mM histidine, 50 to 90 mM NaCl, 2 to 8% sucrose (w/v), 0.5 to 1.5 mM MgCl 2 , 0.005 to 0.05% (w/v) PS-80, 0.05 to 0.15 mM EDTA, and 0.1 to 1.0% ethanol (v/v), and the pH may be 7.0 to 8.0. In another embodiment, when the active ingredient is a nucleic acid, the second chamber is a PBS buffer, for example, PH 7.0 to pH 8.0, 2.0 to 3.5 mM KCl, 1.0 to 2.5 mM KH 2 PO 4 , 125 to 145 mM NaCl, and a solution comprising 7.5 to 9.5 mM Na 2 HPO 4 .
상기 “챔버”는 나노입자의 재료 또는 이를 함유하는 용매를 담기에 적절한 것으로서, 유리, 플라스틱, 종이, 팩 등이 있으나 이에 제한되지 않는다.The "chamber" is suitable for containing a material of nanoparticles or a solvent containing the same, and includes glass, plastic, paper, pack, etc., but is not limited thereto.
이하, 본 발명을 하기 실시예에 의거하여 보다 자세하게 설명하나, 이들은 본 발명을 설명하기 위한 것일 뿐 이들에 의하여 본 발명의 범위가 어떤 식으로든 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail based on the following examples, but these are only for illustrating the present invention and the scope of the present invention is not limited in any way by these.
[실시예][Example]
제조예 1: dioTETA/mPEG-PLA-토코페롤(2k-1.7k)/PLANa(1.7k)을 함유하는 제1 챔버 조성물의 제조 및 나노입자의 형성Preparation Example 1: Preparation of first chamber composition containing dioTETA/mPEG-PLA-tocopherol (2k-1.7k)/PLANa (1.7k) and formation of nanoparticles
(1) 제1 챔버 조성물의 제조(1) Preparation of first chamber composition
1,6-디올레오일 트리에틸렌테트라미드(dioTETA) 20mg을 에탄올 1ml에, 모노메톡시폴리에틸렌클리콜-폴리락타이드-토코페롤 공중합체(mPEG-PLA-토코페롤)(2k-1.7k) 50mg를 90% 에탄올 1ml에, 그리고 폴리락트산나트륨염(PLANa)(1.7k) 50mg를 50% 에탄올에 각각 녹여주었다. dioTETA, mPEG-PLA-토코페롤(2k-1.7k), PLANa(1.7k)를 하기의 표 1의 비율로 섞어준 뒤, 30배의 PBS를 섞어주어 복합 유상액을 제조하였다. 제조된 조성물은 0.22 ㎛ 친수성 필터로 여과시켰다. (표 1 참조)20 mg of 1,6-dioleoyl triethylenetetramide (dioTETA) in 1 ml of ethanol, 50 mg of monomethoxypolyethylene glycol-polylactide-tocopherol copolymer (mPEG-PLA-tocopherol) (2k-1.7k) 90 In 1 ml of % ethanol, and 50 mg of sodium polylactate (PLANa) (1.7k) were dissolved in 50% ethanol, respectively. After mixing dioTETA, mPEG-PLA-tocopherol (2k-1.7k), and PLANa (1.7k) in the ratio shown in Table 1 below, 30 times PBS was mixed to prepare a complex emulsion. The prepared composition was filtered through a 0.22 μm hydrophilic filter. (See Table 1)
[표 1][Table 1]
Figure PCTKR2020018480-appb-I000007
Figure PCTKR2020018480-appb-I000007
(2) 종양용해성 바이러스를 함유하는 제2 챔버 조성물의 제조(2) Preparation of second chamber composition containing oncolytic virus
A195 완충액(10 mM Tris, 10 mM 히스티딘, 75 mM NaCl, 5% 수크로오스 (w/v), 1 mM MgCl2, 0.02% (w/v) PS-80, 0.1 mM EDTA, 0.5% 에탄올 (v/v), pH 7.4)에 분주된 형태의 VQAd CMV Luc 바이러스(ViraQuest, Lot #: 33088)를 1x1010 VP 가 되도록 계수하여 준비하였다.A195 buffer (10 mM Tris, 10 mM histidine, 75 mM NaCl, 5% sucrose (w/v), 1 mM MgCl 2 , 0.02% (w/v) PS-80, 0.1 mM EDTA, 0.5% ethanol (v/v) v), pH 7.4) was prepared by counting VQAd CMV Luc virus (ViraQuest, Lot #: 33088) in the form dispensed to 1x10 10 VP.
(3) 나노입자의 제조(3) Preparation of nanoparticles
상기 제1 챔버 조성물 및 제2 챔버 조성물을 사용 직전에 10 내지 15 초 동안 교반(vortexing)하여 혼합함으로써 나노입자를 형성하였다.Nanoparticles were formed by mixing the first chamber composition and the second chamber composition by vortexing for 10 to 15 seconds immediately before use.
실험예 1: 종양용해성 바이러스를 함유하는 나노입자의 형성 확인Experimental Example 1: Confirmation of formation of nanoparticles containing oncolytic virus
제1 챔버 조성물 및 제2 챔버 조성물을 사용 직전에 혼합함으로써 실시예 1 내지 4의 나노입자가 정상적으로 형성되는지 확인하였다.It was confirmed whether the nanoparticles of Examples 1 to 4 were normally formed by mixing the first chamber composition and the second chamber composition immediately before use.
그 결과, 실시예 1 내지 4 모두 단순 혼합으로도 침전물이 관찰되지 않아, 정상적으로 나노입자가 형성되는 것으로 확인되었다.As a result, in Examples 1 to 4, no precipitate was observed even with simple mixing, and it was confirmed that nanoparticles were normally formed.
실험예 2: 종양용해성 바이러스를 함유하는 나노입자의 세포 내 전달 효율 확인Experimental Example 2: Confirmation of intracellular delivery efficiency of nanoparticles containing oncolytic virus
나노입자의 세포 내 전달 효율을 평가하기 위해, 바이러스 전달효율을 평가하기에 적합한 CAR 발현이 낮은 MDA-MB435 세포를 준비하였다. 제1 챔버 조성물 및 제2 챔버 조성물을 세포 내 주입 직전에 혼합함으로써 실시예 1 내지 4의 나노입자를 형성하고, 바이러스 기준 500moi에 해당하는 양으로 세포에 분주하였다. 15 내지 24시간 동안 추가로 인큐베이션한 후, 세포에 루시페린(luciferin)을 첨가하여 발현하는 루시퍼라아제 양을 측정하였다. 대조군으로는 나노입자가 아닌 바이러스(naked Ad; Ad)를 사용하였다. 그 결과를 도 1에 나타내었다.In order to evaluate the intracellular delivery efficiency of nanoparticles, MDA-MB435 cells with low CAR expression suitable for evaluating the virus delivery efficiency were prepared. The nanoparticles of Examples 1 to 4 were formed by mixing the first chamber composition and the second chamber composition immediately before intracellular injection, and the cells were dispensed in an amount corresponding to 500 moi based on the virus. After additional incubation for 15 to 24 hours, luciferin was added to the cells to measure the amount of luciferase expressed. As a control, a virus (naked Ad; Ad), not nanoparticles, was used. The results are shown in FIG. 1 .
그 결과, 실험된 실시예의 나노입자가 모두 대조군에 비해 세포 내 전달 효율이 높은 것으로 관찰되었다.As a result, it was observed that all of the nanoparticles of the experimental example had high intracellular delivery efficiency compared to the control.
제조예 2: (dioTETA)/mPEG-PLA(2k-1.7k)/PLANa(1.7k)를 함유하는 제1 챔버 조성물의 제조 및 나노입자의 형성Preparation Example 2: Preparation of first chamber composition containing (dioTETA)/mPEG-PLA(2k-1.7k)/PLANa(1.7k) and formation of nanoparticles
(1) 제1 챔버 조성물의 제조(1) Preparation of first chamber composition
dioTETA 20mg를 20 mM 아세트산 나트륨 완충액(pH 4.6) 1ml에, 모노메톡시폴리에틸렌클리콜-폴리락타이드 공중합체(mPEG-PLA)(2k-1.7k) 10mg을 물 1ml에, 그리고 PLANa(1.7k) 10 mg을 물 1mL에 각각 녹여주었다. dioTETA, mPEG-PLA(2k-1.7k), PLANa(1.7k)를 하기의 표 2의 비율로 섞어준 뒤, 0.22 ㎛ 친수성 필터로 여과시켰다. (표 2 참조)20 mg of dioTETA in 1 ml of 20 mM sodium acetate buffer (pH 4.6), 10 mg of monomethoxypolyethylene glycol-polylactide copolymer (mPEG-PLA) (2k-1.7k) in 1 ml of water, and PLANa (1.7k) Each 10 mg was dissolved in 1 mL of water. dioTETA, mPEG-PLA (2k-1.7k), and PLANa (1.7k) were mixed in the ratio shown in Table 2 below, and filtered through a 0.22 μm hydrophilic filter. (See Table 2)
[표 2][Table 2]
Figure PCTKR2020018480-appb-I000008
Figure PCTKR2020018480-appb-I000008
(2) mRNA를 함유하는 제2 챔버 조성물의 제조(2) Preparation of second chamber composition containing mRNA
10 μg CleanCap® FireFly Luciferase mRNA (5-methoxyuridine)(TriLink, Catalog L-7202)를 PBS(PH 7.4, 2.67mM KCl, 1.47mM KH2PO4, 136.9mM NaCl, 8.1mM Na2HPO4)에 용해하여 제2 챔버의 조성물을 준비하였다.Dissolve 10 μg CleanCap ® FireFly Luciferase mRNA (5-methoxyuridine) (TriLink, Catalog L-7202) in PBS (PH 7.4, 2.67 mM KCl, 1.47 mM KH 2 PO 4 , 136.9 mM NaCl, 8.1 mM Na 2 HPO 4 ) Thus, the composition of the second chamber was prepared.
(3) 나노입자의 제조(3) Preparation of nanoparticles
상기 제1 챔버 조성물 및 제2 챔버 조성물을 사용 직전에 10 내지 15 초 동안 교반(vortexing)하여 혼합함으로써 나노입자를 형성하였다.Nanoparticles were formed by mixing the first chamber composition and the second chamber composition by vortexing for 10 to 15 seconds immediately before use.
실험예 3: mRNA 나노 입자의 형성 확인Experimental Example 3: Confirmation of the formation of mRNA nanoparticles
제1 챔버 조성물 및 제2 챔버 조성물을 사용 직전에 혼합함으로써 실시예 5 내지 8의 나노입자가 mRNA와 결합하며 형성되는지 확인하였다. mRNA complexation은 agarose gel retardation assay(1.5% agarose; DyneGelSafe Kit)를 이용하여 측정하여 결과를 도 2에 나타내었다. 그리고 나노입자 형성 확인을 위하여 DLS를 사용한 입자크기 측정 결과를 도 3에 나타내었다. 실시예 5 내지 8 모두 free mRNA와 비교하여 gel retardation assay 결과와 DLS 결과를 바탕으로 mRNA complex 나노입자가 형성되는 것으로 확인되었다.By mixing the first chamber composition and the second chamber composition immediately before use, it was confirmed whether the nanoparticles of Examples 5 to 8 were formed by binding to mRNA. mRNA complexation was measured using agarose gel retardation assay (1.5% agarose; DyneGelSafe Kit), and the results are shown in FIG. 2 . And the particle size measurement results using DLS to confirm the formation of nanoparticles are shown in FIG. 3 . In all of Examples 5 to 8, it was confirmed that mRNA complex nanoparticles were formed based on gel retardation assay results and DLS results compared with free mRNA.
실험예 4: mRNA를 함유하는 나노입자의 세포 내 전달 효율 확인Experimental Example 4: Confirmation of intracellular delivery efficiency of nanoparticles containing mRNA
생성된 나노입자의 세포 내 전달 효율을 평가하기 위해, A549 human lung cancer 세포를 준비하였다. 제1 챔버 조성물 및 제2 챔버 조성물을 세포 내 주입 직전에 혼합함으로써 실시예 6 또는 8에 해당하는 나노입자를 형성하고, 96 well 기준 250 ng mRNA에 해당하는 양으로 세포에 분주하였다. 6시간 동안 추가로 인큐베이션한 후, 세포에 루시페린 (luciferin)을 첨가하여 발현하는 루시퍼라아제 양을 측정하였다. 대조군으로는 commercial reagent인 TransIT®-mRNA Kit(Mirus Bio)를 사용하였다. 그 결과를 도 4에 나타내었다. 그 결과, 실험된 실시예의 나노입자가 대조군과 유사한 수준의 세포 내 전달 효율을 보이는 것으로 관찰되었다.To evaluate the intracellular delivery efficiency of the generated nanoparticles, A549 human lung cancer cells were prepared. Nanoparticles corresponding to Examples 6 or 8 were formed by mixing the first chamber composition and the second chamber composition immediately before intracellular injection, and were dispensed into cells in an amount corresponding to 250 ng mRNA in 96 wells. After additional incubation for 6 hours, the amount of luciferase expressed by adding luciferin to the cells was measured. As a control, a commercial reagent, TransIT ® -mRNA Kit (Mirus Bio) was used. The results are shown in FIG. 4 . As a result, it was observed that the nanoparticles of the experimental example showed a similar level of intracellular delivery efficiency as the control.
실험예 5: mRNA를 함유하는 나노입자의 안정성에 따른 세포 내 전달 효율 비교Experimental Example 5: Comparison of intracellular delivery efficiency according to the stability of nanoparticles containing mRNA
나노입자의 안정성에 의해 투여 직전에 키트로 제조된 나노입자가 세포 내 전달 효율이 더 높음을 확인하기 위해, HepG2 세포를 준비하여 가속 시험으로서 세포 내 전달 효율을 비교하였다. 제1 챔버 조성물 및 제2 챔버 조성물을 세포 내 주입 직전에 혼합함으로써 실시예 6에 해당하는 나노입자를 형성하고, 96 well 기준 250 ng mRNA에 해당하는 양으로 세포에 분주하였다. 비교군으로 동일한 방식으로 제조한 나노입자와, 통상적으로 transfection agent로 사용되는 L3K를 이용하여 제조한 나노입자를 42℃에서 1 시간 보관한 것을 사용하고, 동일하게 세포에 분주하였다. 15시간 동안 추가로 인큐베이션한 후, 세포에 루시페린(luciferin)을 첨가하여 발현하는 루시퍼라아제 양을 측정하였다. 그 결과를 도 5에 나타내었다. 그 결과, 제조 후 가속 시험에 의해 안정성이 저하되어 세포 내 전달 효율이 급격히 감소하는 것을 확인하였다.In order to confirm that the nanoparticles prepared with the kit have higher intracellular delivery efficiency just before administration due to the stability of the nanoparticles, HepG2 cells were prepared and the intracellular delivery efficiency was compared as an accelerated test. Nanoparticles corresponding to Example 6 were formed by mixing the first chamber composition and the second chamber composition immediately before intracellular injection, and were dispensed into cells in an amount corresponding to 250 ng mRNA in 96 wells. As a comparison group, nanoparticles prepared in the same manner and nanoparticles prepared using L3K, which is usually used as a transfection agent, stored at 42° C. for 1 hour were used, and the same was dispensed into cells. After additional incubation for 15 hours, luciferin was added to the cells to measure the amount of luciferase expressed. The results are shown in FIG. 5 . As a result, it was confirmed that the stability was lowered by the accelerated test after preparation, and the intracellular delivery efficiency was rapidly reduced.

Claims (8)

  1. 양친성 블록 공중합체, 양이온성 화합물 및 폴리락트산염을 포함하는 제1 챔버; 및a first chamber containing an amphiphilic block copolymer, a cationic compound, and a polylactate; and
    핵산, 폴리펩티드, 바이러스 또는 이들의 조합으로부터 선택된 유효성분을 포함하는 제2 챔버;를 포함하는,A second chamber containing an active ingredient selected from a nucleic acid, a polypeptide, a virus, or a combination thereof;
    나노입자 조성물 제조용 키트.A kit for preparing a nanoparticle composition.
  2. 제1항에 있어서, 세포 내 유효성분을 전달하는 나노입자를 형성하기 위한 것인, 나노입자 조성물 제조용 키트.The kit for preparing a nanoparticle composition according to claim 1, for forming nanoparticles that deliver an intracellular active ingredient.
  3. 제1항에 있어서, 상기 제1 챔버 및 제2 챔버로 이루어진 군 중에서 선택된 하나 이상은 추가의 용매를 더 포함하는 것인, 나노입자 조성물 제조용 키트.The kit of claim 1, wherein at least one selected from the group consisting of the first chamber and the second chamber further comprises an additional solvent.
  4. 제3항에 있어서, 상기 용매는 수성 용매, 수혼화성 용매 또는 이의 혼합물인, 나노입자 조성물 제조용 키트.The kit according to claim 3, wherein the solvent is an aqueous solvent, a water-miscible solvent, or a mixture thereof.
  5. 제1항에 있어서, 상기 제2 챔버는 pH 조절제, 무기염, 당류, 계면활성제, 킬레이트제 또는 이들의 조합을 더 포함하는 것인, 나노입자 조성물 제조용 키트.The kit of claim 1, wherein the second chamber further comprises a pH adjusting agent, an inorganic salt, a saccharide, a surfactant, a chelating agent, or a combination thereof.
  6. 제1항에 있어서, 상기 양친성 블록 공중합체의 양은 상기 양이온성 화합물 1 중량부를 기준으로 0.01 내지 50 중량부인, 나노입자 조성물 제조용 키트.According to claim 1, wherein the amount of the amphiphilic block copolymer is 0.01 to 50 parts by weight based on 1 part by weight of the cationic compound, the nanoparticle composition preparation kit.
  7. 제1항에 있어서, 상기 폴리락트산염의 양은 상기 양이온성 화합물 1 중량부를 기준으로 0.1 내지 100 중량부인, 나노입자 조성물 제조용 키트.The kit for preparing a nanoparticle composition according to claim 1, wherein the amount of the polylactic acid salt is 0.1 to 100 parts by weight based on 1 part by weight of the cationic compound.
  8. 제1항에 있어서, 상기 제1 챔버에서 상기 양친성 블록 공중합체, 양이온성 화합물, 및 폴리락트산염의 혼합물은, 혼합 후 친수성 필터로 1회 이상 여과된 것인, 나노입자 조성물 제조용 키트.The kit for preparing a nanoparticle composition according to claim 1, wherein the mixture of the amphiphilic block copolymer, the cationic compound, and the polylactate in the first chamber is filtered at least once with a hydrophilic filter after mixing.
PCT/KR2020/018480 2019-12-20 2020-12-16 Kit for preparing nanoparticle composition for drug delivery, comprising polylactic acid salt WO2021125805A1 (en)

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CA3162374A CA3162374A1 (en) 2019-12-20 2020-12-16 Kit for preparing nanoparticle composition for drug delivery, comprising polylactic acid salt
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